Recent Progress in the Synergistic Combination of Nanoparticle-Mediated Hyperthermia and Immunotherapy for Treatment of Cancer

Abstract

Immunotherapy has demonstrated great clinical success in certain cancers, driven primarily by immune checkpoint blockade and adoptive cell therapies. Immunotherapy can elicit strong, durable responses in some patients, but others do not respond, and to date immunotherapy has demonstrated success in only a limited number of cancers. To address this limitation, combinatorial approaches with chemo- and radiotherapy have been applied in the clinic. Extensive preclinical evidence suggests that hyperthermia therapy (HT) has considerable potential to augment immunotherapy with minimal toxicity. This progress report will provide a brief overview of immunotherapy and HT approaches and highlight recent progress in the application of nanoparticle (NP)-based HT in combination with immunotherapy. NPs allow for tumor-specific targeting of deep tissue tumors while potentially providing more even heating. NP-based HT increases tumor immunogenicity and tumor permeability, which improves immune cell infiltration and creates an environment more responsive to immunotherapy, particularly in solid tumors.

Graphical Abstract

Nanoparticle-based hyperthermia therapy (HT) demonstrates significant potential as an adjuvant to immunotherapy. Nanoparticle-based HT allows for tumor-specific targeting of deep tissue tumors while providing even heating to address limitations of clinical HT. NP-based HT can increase tumor immunogenicity and tumor permeability, creating an environment that is more responsive to immunotherapy through improved immune cell activation and infiltration.

1. Introduction

In recent years, advances in immunotherapy-based treatment have led to great strides in cancer therapy. The immune system has long been known to play a role in the treatment of cancer.^[1] Over the last decade, our increased understanding of how cancer cells evade the immune system has driven the development of clinically successful immunotherapies such as the use of check point inhibitors and antigen-specific adoptive cellular therapy.^[2,3] In 2010, sipuleucel-T was approved by the FDA for treatment of prostate cancer as the first autologous cancer vaccine based-immunotherapy.^[4] The first immune checkpoint blockade (ICB) therapy was approved for the treatment of metastatic melanoma by the FDA in 2014, followed by approval of ICB therapy in Europe in 2015.^[3,5] China approved its first ICB-based immunotherapy for the treatment of cancer in 2018.^[6] Immunotherapy has been shown to provide a significantly longer and more durable response than traditional chemo- or radiotherapies.^[7] Immunotherapies have also proven effective in treating the heavy tumor burden of some bulky tumors and metastatic cancer, areas traditional chemo-, radio-, and targeted therapies have failed to address.^[8–10] Additionally, immunotherapies have demonstrated more tolerable toxicity than traditional therapy.

Despite these achievements, immunotherapy has not demonstrated efficacy in treating many forms of cancer. Furthermore, patients that are treated with immunotherapy often show varying responses within cohorts.^[11] The varying response rates may be attributed to a lack of patient specificity to elicit an immune response, cancer-specific mechanisms employed to evade immune surveillance, and inability of activated immune cells to efficiently access tumors. To address these limitations, the combination of immunotherapy with other therapeutic interventions that increase tumor immunogenicity such as chemo-, radio-, and hyperthermia therapy (HT) have been extensively investigated.^[12] Of these combinatorial approaches, HT is particularly suited as an adjuvant therapy to immunotherapy because it specifically addresses limitations of current immunotherapy approaches with minimal toxicity.

HT, which involves raising the temperature of the targeted tumor tissue to 39–45 °C, has become a viable strategy to treat cancer. HT causes thermally induced metabolic changes that can lead to apoptosis and has shown to trigger an immune response.^[13] Although HT has not yet been accepted as a front line therapy, it has demonstrated the ability to debulk tumors, and has been shown in many phase II and III clinical trials to successfully augment chemo- and radiotherapy.^[14,15] However, to date HT has only received clinical approval in Europe for limited applications.^[16,17] While not yet broadly investigated clinically, the cellular and extracellular effects of HT on tumor tissue make it a good candidate for adjuvant therapy to immunotherapy. HT enhances the immune response through several mechanisms, including modulation of surface receptors and pro-inflammatory cytokines, increased release of antigens, proliferation of immune cells, and increased activation and migration of immune effector cells, all of which work in concert to increase cytotoxicity against malignant cells.

A drawback of current clinically investigated approaches to HT is the lack of tissue specificity, which can lead to damage in healthy tissue, and limited penetration depth which restricts the types of tumors that can be treated with this modality. However, emerging nanotechnology platforms provide means to facilitate tumor-specific targeted HT at any depth in the body, opening up new opportunities to maximize the synergistic combination of HT with immunotherapy.^[18,19] This progress report will provide a brief summary of current immunotherapy approaches, discuss the various approaches used to facilitate HT, and describe the mechanisms with which HT induces an immune response. Finally, this report will elucidate the potential synergy between HT and immunotherapy and highlight recent advancements in nanoparticle-based magnetic HT (MHT) and photothermal therapy (PTT) in combination with immunotherapy.

2. The Immune System and Its Role in Pathogenesis and Treatment

Immunotherapy has become the latest addition to the pillars of cancer therapy, which include surgery, chemotherapy, radiotherapy, and targeted therapy. It has long been recognized that the immune system plays a critical role in the development and potential treatment of cancer.^[20] Over the last 20 years, the concept of immunosurveillance, which was first introduced by Paul Erlich in 1909,^[21] has been adapted into a theory termed immunoediting to better describe the complex role the immune system plays in cancer development.^[3] Responsible for both constraining and promoting tumor development, cancer immunoediting progresses through three phases: elimination, equilibrium, and escape. Elimination represents the successful rejection of evolving tumors by the innate and acquired immune system, and characterizes the endpoint of immunoediting. If the tumor cells are not completely eradicated, the cells may enter the equilibrium state where the immune system controls tumor growth and the cell may acquire a state of quiescence. Escape represents the evasion of the immune system by tumor cells that leads to proliferation and the development of clinically recognized tumors. However, these phases are not separate but represent a continuum and complex interplay between tumor growth and immune response. Through the process of immunoediting, tumor immunogenicity is edited and an immunosuppressive tumor microenvironment (TME) can arise that recruits specific immune cells to facilitate tumor growth and progression. A deeper understanding of the underlying mechanisms of immunoediting and the interplay of a host of ligands and receptors associated with tumor and immune cells has provided the basis for clinical immunotherapy (Figure 1).

Figure 1.

Immune-regulation in the TME is dictated by checkpoints located on immune cell membranes. The check point receptors on T cells interact with their ligands on the surface of APCs or tumor cells, and provide either stimulatory (green) or inhibitory (red) signals between the two cells. Checkpoint blockade-based immunotherapy acts by blocking the inhibitory axes. APC, antigen-presenting cell; Treg, regulatory T cell; TAM, tumor-associated macrophage; NK, natural killer.

2.1. Immune Cells and Their Therapeutic Implications

2.1.1. T Cells

T cells play a critical role in immunoediting and have significant therapeutic potential. There are various types of T cells involved in immunosurveillance. Regulatory T cells (Tregs) co-express CD4, CD25, and transcription factor FoxP3, while effector and memory T cells express CD4 or CD8.^[22,23] Key drivers of T cell expansion are tumor-specific antigens (TSAs), which are only expressed in tumor cells, and tumor-associated antigens (TAAs), which are expressed in normal and tumor cells. Tumor infiltrating lymphocytes (TILs, T cells and B cells) may specifically target TSAs and an increase in the presence of cytotoxic TILs corresponds with increased patient survival.^[24] The immune system can recognize TAAs and generate both tumor specific CD4⁺ and CD8⁺ T cells along with antibodies against TAAs. Because cancer originates from normal cells which also present TAAs, the mechanisms that lead to self-tolerance can also cause poor anti-tumor response from the immune system.

A key component of immune regulatory mechanisms is Tregs. Tregs are attracted to tumor tissue by tumor cell and macrophage secretion of factors including C-C motif chemokine ligands 5, 20, and 22 (CCL5/20/22).^[25] In the tumor microenvironment, Tregs become activated by TAAs or self-antigens released by dying tumor cells. Tregs are capable of suppressing a number of immune cells including CD8⁺ T, natural killer (NK), B, and antigen-presenting cells (APCs).^[26,27] Evidence suggests an increased presence of Tregs may cause inhibition of efficacious antitumor immune response through the suppression of cytotoxic immune cells. The proportion of peripheral Tregs in the blood has been shown to be significantly higher in cancer patients for a wide array of cancers. However, mechanisms that regulate Tregs are complex and context-dependent given that in some cancers, an increase in Tregs is correlated with better prognosis.^[28]

CD8⁺ effector T cells are believed to mitigate local tumor growth through direct cell killing and through release of effector cytokines including interferon gamma (IFN-γ) or tumor necrosis factor (TNF). Therefore, an increase in the number of CD8⁺ effector T cells within a tumor is associated with better patient prognosis.^[29] However, the potency of CD8⁺ T cells is controlled by the balance between co-inhibitory and co-stimulatory signals associated with the immune checkpoints.^[30] In addition, CD8⁺ memory T cells have shown to protect against metastatic recurrence. This suggests that CD8⁺ memory T cells are effective in controlling the migration of metastatic tumor cells.^[31]

An increased understanding of T cell immunoediting mechanisms has led to therapeutic approaches including variations on adoptive T cell transfer therapy and immune checkpoint therapy that focuses on depletion of Tregs. Adoptive T-cell transfer involves the isolation, expansion, activation of TILs ex vivo.^[32] The concept behind this approach relies on the removal of TILs from the immunosuppressive microenvironment and subsequent activation which mitigates tumor induced T-cell dysfunction. Several variations of this approach have manifested over the years.^[33] One promising example is the genetic manipulation of immune cells to enhance their ability to specifically target tumor antigens. Autologous T cells can be modified with conventional T cell receptors (TCRs) or chimeric antigen receptor (CAR).^[34] CARs specifically recognize TSAs, and CAR T cells (CARTs) can initiate direct killing of tumor cells. CART therapy has generated promising results in clinical trials.^[35] The reduction of immune inhibitory Tregs may be an alternative approach to improving antitumor immune activity. The cytotoxic T-lymphocyte antigen 4 (CTL-4) expressed on the surface of Tregs can be targeted by anti-CTL-4 antibodies as a checkpoint blockade therapy.^[36]

2.1.2. Natural Killer Cells

NK cells are capable of spontaneously lysing tumor cells without prior activation and play an important role in immunosurveillance.^[37] Studies have shown that high intratumoral NK cell infiltration in solid tumors, including lung, gastric, colorectal, and head and neck cancers, is associated with better prognosis in patients.^[38,39] NK cell cytolytic activity is controlled and activated by a variety of cell surface receptors known as the natural cytotoxicity receptors (NCRs), which have high affinity for ligands that are primarily expressed by cells that are ‘stressed’.^[40] NK cells also express receptors that can act as inhibitory or stimulatory receptors such as killer Ig-like receptors (KIR) and CD94/NKG2 specific to the major histocompatibility complex (MHC) class I molecules.^[3] A consequence of this is that NK cells lyse cells that have lost MHC I molecules which is common in tumor cells.^[41] NK cells utilize this mechanism to kill cells that have successfully escaped regulation by CD8⁺ T cells. It has been reported that NK cells may use this mechanism to kill cancer stem cells responsible for metastasis. Dendritic cells, both immature and mature have been shown to activate NK cells and increase their antitumor activity through release of cytokines such as IFN-α.^[42]

Tumor cells are capable of escaping NK cells by either suppressing NK cell function or by immunoediting of poorly immunogenic tumor cells.^[2] Suppression of NK cell function can be achieved through the downregulation of NK-attracting chemokines, such as CXCL2 in the TME or through inhibition of the cytolytic function of NK cells by mediators such as TGF-β which reduce the surface expression of NCRs.^[43] NK cells can lose potency by exhaustion through continuous exposure to some target antigens. In addition, the hypoxic TME can act as a suppressor by significantly reducing the expression of NCRs.^[44] Immunoediting of tumors can reduce NK potency by reducing the expression of ligands such as NKG2D on tumor cells.^[45] Tumor cells are also capable of increasing expression of MCH I which can inhibit NK cytotoxic function. Tregs are capable of regulating NK cells through interleukin-2 (IL-2) competition.^[46] The reduced availability of IL-2 decreases NK cell proliferation and negatively effects NK cell cytotoxicity. In certain cancers, the tumor has a direct effect on the NK cell phenotype, reducing cytolytic activity and causing an increase in VEGF expression. These NK cells exhibit angiogenic characteristics and promotes tumor progression. Studies have suggested these altered NK cells directly reduce the number of T cells in the TME further inhibiting an anticancer immune response.^[47]

To overcome the mechanisms employed by tumors to suppress NK cell function, many strategies have been investigated, including the utilization of adoptive transfer of NK cells through genetic modification of NK cells to increase resilience and cytolytic activity, and the use of immune stimulants.^[48] Clinically, NK cells are being investigated for applications similar to that of adoptive T cells transfer. Ex-vivo expansion of alloreactive NK cells followed by infusion alone or in combination with IL-2 has led to beneficial responses in some patients. CAR NK cell therapy is also showing promising result in early clinical trials, particularly when used in combination with chemotherapy.^[49] The use of anti-KIR antibodies to manipulate activating or inhibitory receptors on NK cells has shown anti-tumor effects in mouse models and clinical trials of this approach are currently on going.^[50]

2.1.3. Dendritic Cells

Dendritic cells (DCs) are an important component of antitumor immune response because they are capable of cross-presenting antigens to CD4⁺ and CD8⁺ T cells.^[51] There is evidence to suggest DCs can act as a direct cytotoxic effector cell, however, they seem to serve a more prominent role as an antigen presenting cell (APC).^[52] Mature DCs are required to provide efficient antigen presentation to stimulate T cells, and the TME regulates the maturation of DCs. In tumors, DCs are often found in their immature or tolerogenic phenotype due to factors released by the tumor or tumor associated macrophages (TAMs), including VEGF, IL-8, and IL-10.^[53] Immature DCs release factors such as IL-10, TGF-β, and intoleamine-2,3-dioxygenase, which leads to poor stimulation of T cells,^[54] and immature DCs spur the expansion of Tregs.^[55] Not only can immature DCs suppress an antitumor immune response, they can actively promote tumor growth through production of proangiogenic factors and increased endothelial cell migration.^[56] Conversely, mature DCs suppress proangiogenic factors,^[57] and release high amount of IL-12, leading to T cell activation.^[58] Infiltration by mature DCs into primary tumors is associated with a reduction in metastases.^[59] However, tumors are able to reprogram mature DCs into an immunosuppressive and proangiogenic phenotype. DCs are capable of infiltrating a wide variety of tumors, but their effect on patient outcomes is not conclusive.

Extensive research has been undertaken to develop personalized cancer vaccines utilizing patient derived DCs that are manipulated ex-vivo.^[60] These approach typically utilize isolated monocytes or hematopoietic stem and progenitor cells from peripheral blood, which are differentiated using recombinant cytokines, stimulated to induce maturation, and functionalized with TAAs of various types. This process has been used to develop DC-based vaccines that have been tested in numerous clinical studies.^[61] While DC-based vaccines have demonstrated acceptable safety and immunogenic responses in clinical trials, clinical response has been disappointing. This has been attributed to insufficient antigen presentation, capacity for migration, and cytokine release. Despite these limitations, new research is expanding our understanding of the role DCs play in antitumor immune response and is opening new approaches to develop efficacious DC-based vaccines.^[62,63]

2.1.4. Macrophages

Tumor-associated macrophages (TAMs) comprise a sizeable fraction of non-tumor derived cells in tumors. In nonmalignant tissue, macrophages act to restore functional homeostasis by phagocytosis of apoptotic cells following injury and play a role in immune surveillance.^[64] In a resting state, macrophages monitor their local environment for chemokines indicative of trauma, infection, or malignancy. Macrophages responding to injury and infection are activated to a pro-inflammatory (M1) state characterized by secretion of pro-inflammatory cytokines, enhanced phagocytosis, and antigen presentation to immune cells, leading to antigen specific T cell response.^[65,66] To limit damage to normal tissue, macrophages are activated to an anti-inflammatory M2 state that restricts immune response and promotes tissue recovery. Tumors subvert the normal function of M2 TAMs by secreting a host of factors including chemokines, colony stimulating factors (CSFs), and transforming growth factor beta (TGF-β) that recruit macrophages to the TME and stimulate their proliferation.^[67,68] Tumors then co-opt the secretory products of M2 TAMs to produce an anti-inflammatory and immunosuppressive TME environment that promotes tumor growth and survival. TAMs promote tumor immune evasion through interaction of signal regulatory protein alpha (SIRP-α) with CD47 on tumors. Additionally, TAMS release chemokines including CCL5, CCL20, and CCL22 which recruit Tregs. Pro-tumorigenic M2 TAMs also produce factors that foster tissue degradation and angiogenesis, facilitating tumor cell invasion and metastasis. In addition, recent reports indicate that pro-tumorigenic TAMs promote tumor resistance to chemo-, radio-, and immunotherapy.^[69–73] The association between M2 state and tumor progression strongly suggests that targeting the interaction between TAMs and tumor cells could improve clinical outcomes. Preclinical evaluation of macrophage colony-stimulating factor (M-CSF) binding to its receptor CSF-R1 has shown to be instrumental in the polarization of macrophages to the M2 state and blockages of this interaction using small molecular inhibitors has shown promise in animal models.

2.2. Immune Checkpoints and Their Therapeutic Implications

Naive T cells require at least two signals to become fully activated upon recognition of antigens presented on the surface of APCs or tumors. The first signal is transduced through the interaction of TCRs with peptide-MHC complexes and is antigen specific. The second signal is antigen independent and is transduced by specific co-receptors on T cells belonging to the B7/CD28 protein family and binds to CD80/86 on APCs or tumor cells.^[74] Co-stimulatory receptors such as CD28 promote the activation of T cells, while inhibitory receptors including programmed cell death-1 (PD-1), CTLA-4 inhibit T cell function and are termed ‘immune checkpoint molecules’.^[75] These inhibitory receptors are intended to prevent inappropriate immune reactions by limiting the extent and duration of immune response. TCR stimulation by programmed death ligands 1 or 2 (PD-L1/2) present on APCs or tumor cells leads to a blockade of Akt signaling which in turn causes decreased cytokine production, as well as T cell proliferation and survival. Cytokines produced after T cell activation lead to a negative feedback loop through upregulation of PD-1 ligands.^[76] PD-1 is constitutively expressed on a subset of thymic T cells and is upregulated on activated NK, T and B cells, monocytes and DCs, and CD4+ follicular helper T cells. The PD-1/PD-L1 pathway represents a critical T cell resistance mechanism in tumors. PD-L1 is expressed by a variety of epithelial and hematological malignancies and PD-1 is significantly upregulated in cancer-specific T cells, suggesting these cancers may use the PD-1/PD-L1 signaling pathway to escape antitumor immune response and facilitate tumor progression. Studies have shown that PD-L1 induces resistance to T cell mediated killing and inhibits apoptosis induced by antigen specific T cells.^[77,78] Furthermore, PD-L1 expression on tumors is correlated with poorer patient outcomes in a variety of cancers.^[79] These mechanisms may be responsible for the lack of tumor response in numerous trials evaluating adoptive cell therapy.

Targeting immune check point receptors represents a major breakthrough in clinical immunotherapy. The first FDA approved ICB therapy was Ipilimumab, a human IgG1 antibody targeting CTLA-4, and was approved in 2011 for the treatment of metastatic melanoma.^[80] Significantly, an analysis of the 10 years of various Ipilimumab trials showed that ~20% of patients achieved long-term remission, suggesting a prolonged, durable response. In 2014, the FDA approved nivolumab and pembrolizumab, both human IgG4 antibodies against PD-1, as the second immune checkpoint therapies for treatment of malignant melanoma.^[81,82] Nivolumab has subsequently been approved for the treatment of relapsed squamous non-small cell lung cancer.^[83] Furthermore, the combination of nivolumab with CTLA-4 blockade has shown impressive results in recent studies against metastatic melanoma.^[84] Pembrolizumab has shown to be more effective than ipilimumab as first-line therapy against metastatic melanoma. Pembrolizumab has also shown some efficacy against solid tumors including lung, head and neck, triple-negative breast cancer, and renal cell carcinoma.^[85] The FDA has recently granted accelerated approval for three PD-L1 inhibitors for use in a various cancers, including non-small cell lung cancer, Merkel cell carcinoma, urothelial cancer, and triple negative breast cancer.^[86] There are currently ongoing phase II/III trials for the FDA approved PD-L1 inhibitors atezolizumab, durvalumab, and avelumab. In addition, there are several new PD-L1 inhibitors currently in the developmental pipeline.

3. Hyperthermia Therapy as Immunotherapy

HT refers to use of elevated temperatures of the whole body and/or local tumor tissue and the secondary effects to treat cancer. The use of local HT to treat cancer dates back to Ancient Egypt where the use of cautery to locally destroy tumors was widely used by 2000 BC. In the late 19^th century, the observation that infections and associated fever episodes led to cancer remission not only inspired the idea that the immune system plays a critical role in cancer therapy, but also spurred the investigation of hyperthermia as a legitimate treatment. William Coley’s famous trials in the 1890’s showed the 5-year survival rate associated with treatment by bacterial extracts known as Coley’s toxins, were directly related to achieved body temperatures. In his studies, a fever over 38.5 °C, led to survival past 5 years for 60% of his patients, while only 28% survived 5 years if fever remained below 38.5 °C.^[87] While these results don’t make clear whether the improved outcomes were caused by high fever, or from the increased immune response associated with increased fever, it did generate interest in the idea of systemic hyperthermia as a cancer therapy. Direct evidence of the effect of hyperthermia was documented by William Mayo in 1913. In his studies, tumor dissemination post-surgery was significantly reduced and cure rates were higher when heating cervical tumors with a cautery prior to vaginal hysterectomy. Specifically, the improved outcomes were only observed if a time delay was given between the heating of the tumor and surgical removal. This implied that there was a protective measure provided by local hyperthermia that we now assume is the activation of antitumor immune response.

3.1. Methods for Inducing Hyperthermia

Application of HT typically utilizes temperatures in the fever range (mild hyperthermia) or temperatures above the fever range that can ablate tumors. Hyperthermia can be achieved through various methods. Historically, whole body HT used to treat metastatic disease included exposure to infrared light and the use of heated water baths.^[88] However, these approaches are limited to treatment of tumors near the surface of the skin due to limitation in penetration depth. With these approaches, it is also difficult to accurately control local temperatures, both spatially and in magnitude. Local/regional clinical HT is mainly delivered by microwaves, radiofrequency waves, or ultrasound.^[89] The use of low frequency electromagnetic waves such as microwaves and radiofrequency waves can penetrate in to the body up to 15 cm, but are difficult to focus. Higher frequencies are easier to focus, but tissue absorption at higher frequencies doesn’t allow deep tissue penetration. Due to these limitations, electromagnetic wave-based techniques are mostly limited to superficial tumors or broader regional heating of deeper tissue.^[90] Ultrasound, which relies on mechanical waves, is able to generate heat through mechanical friction at depths of up to 20 cm, allowing for deep tissue treatment.^[91] High intensity focused ultrasound (HIFU), is FDA approved for the treatment of cancer and is capable of both ablative applications and lower intensity mild hyperthermia.^[92] The disadvantages of ultrasound-based techniques are high bone absorption and the inability to penetrate through tissues that contain air, as in the respiratory and gastrointestinal tract.

Recent advances in nanotechnology have provided the ability to specifically target tumor tissue and facilitate non-invasive HT. The two main approaches are nanoparticle-based MHT and PTT. Localized heating with MHT relies on delivery of magnetic nanoparticles (NPs) to tumor tissue followed by the application of an alternating magnetic field (AMF) to produce heat through hysteresis losses which are dissipated as thermal energy.^[93] NP-based PTT relies on photoconversion nanomaterials to convert light energy into heat.^[94] The heating efficiency of NPs is assessed through the specific heat absorption rate (SAR) which experimentally is measured using calorimetry (MHT and PTT), magnetometric methods (MHT), or infrared/fluorescence thermometry (MHT and PTT) which have been discussed in detail in recent review articles.^[93,95] NP-based PTT and MHT can provide targeted HT via enhanced permeability and retention (EPR) mechanisms or molecular targeting, and many formulations of NPs designed for MHT and PTT can be visualized by clinical imaging technology. NP-based MHT and PTT are currently being assessed in clinical trials for treatment of glioblastoma (MHT) and prostate cancer (MHT and PTT).^[96,97]

3.1.1. Photothermal Therapy

NP-based PTT involves irradiation of NPs with light, usually in the near infrared (NIR) range (700–900 nm) to facilitate hyperthermia. Good photothermal NP agents must facilitate strong absorbance of NIR, efficiently transfer absorbed light into heat, and be non-toxic.^[98] NPs of various materials and geometries have been utilized as photosensitizers (PSs) in the design of PTT agents. Materials include small organic dyes, gold, copper, graphene, polymers, carbon nanotubes, iron oxide, silver and platinum, however gold, copper, and graphene are the most common materials used.^[94,99] NP geometries include nanoshells, nanorods, nanostars, and nanocages. Since PTT is applied with NIR wavelengths, its application is restricted to penetration depths of ~1 cm. This limitation requires that the tumor is either subcutaneous, surgically exposed, or accessible by catheter. The majority of research on PTT has been performed in preclinical in vivo models, however, PTT has begun to be investigated in clinical trials as a cancer therapy.^[100]

The efficiency of NP-based PTT agents is dictated by the choice of PS, the core morphology, and the surface properties of the NP.^[101] Organic dyes and carbon-based materials function as PSs for PTT by elevating an electron from its ground state to an excited state upon absorbance of a photon followed by internal relaxation to the lowest vibrational level of the excited state. Further relaxation of the molecule results in the transfer of energy to the surrounding environment as heat.^[102,103] Noble metal and semiconductor NPs used as PSs respond quite differently to incident light. In these materials, light induces the coherent oscillation of conduction electrons relative to the lattice in a phenomena known as localized surface plasmon resonance (LSPR). The oscillation frequency depends on the size, shape and composition of the NPs. Upon decay of the surface plasmon oscillation, energy is transferred nonradiatively as heat or by radiating energy as light to facilitate PTT, and photoacoustic and optical imaging.^[104,105] In vivo applications require the LSPR frequency to be tuned to the NIR range to allow for deeper tissue penetration. For gold nanoparticles, the LSPR frequency can be tuned by adjusting the shape of the NP. Increasing the aspect ratio of the NP into nanorods or nanocages shifts the LSPR frequency from visible light to the NIR range. The formation of gold nanoshells leads to pinholes in the shell, which increase with decreasing shell thickness. An increase in the number of pinholes with reduced shell thickness leads to a red-shift of the LSPR peak to the NIR range.^[106,107] Gold nanorods and hollow gold nanoshells serve as the predominant NP used for PTT therapy because the LSPR frequency can be easily tuned to the NIR range.

3.1.2. Magnetic Hyperthermia Therapy

MHT relies on magnetic NPs that convert magnetic energy to heat energy in the presence of an AMF. The efficiency of this conversion process can be tuned by modulating particle size, composition, and shape.^[93] The use of AMF to activate NPs is a significant advantage because the AMF has no penetration depth limitation. In addition to providing a mechanism for heating, the magnetic properties of the NPs can facilitate magnetic targeting of tumors, further improving tumor specificity.^[108] Iron oxide is the predominant material used for NP-based MHT, although various compositions using other metal dopants have been investigated.^[109] Various polymer coatings are utilized to passivate magnetic NP surfaces and provide steric stabilization. Polyethylene glycol (PEG) is commonly used as a polymer coating due to its relative inertness and non-toxic safety profile in vivo. Many formulations of iron oxide-based magnetic NPs have previously been approved for clinical MR imaging applications indicating the overall safety profile of these NPs.^[110]

Many factors influence the conversion efficiency of magnetic energy to heat through magnetic loss of the NP, including size, composition, shape, and surface coating.^[93] The amount of heat produced by magnetic NPs is approximately equal to the area of the hysteresis loop during a complete cycle of the magnetic field and the efficiency of this process is evaluated by the specific heat absorption rate.^[111,112] Size, composition, shape and surface coatings have a direct effect on SAR because these factors can affect magnetic susceptibility, saturation magnetization (M_S), crystalline anisotropy (K), and relaxation time.^[113,114] An increase in the ratio of magnetic susceptibility/M_S leads to greater heating efficiency due to the reduced K and spin disordering on the NP surface. Magnetic susceptibility/M_S increases as particle size increases until a maximum value is reached which is dictated by particle composition and morphology.^[115] The SAR value is inversely proportional to the size distribution indicating that highly monodisperse NPs are more efficient at heat conversion. Composition is another means to significantly alter NP magnetic properties. For ferrite-based NPs, the position of metal atoms in the octahedral and tetrahedral sites effects magnetic properties. By doping ferrite-based NPs with transition metal cations including Mn²⁺, Ni²⁺, Co²⁺, or Zn²⁺, M_S can be tailored to increase the SAR value.^[116,117] In addition to composition, NP shape is another major factor utilized to improve SAR. When comparing cubic NPs with spherical NPs of the same size and composition, it has been shown that the cubic NP exhibited significantly higher SAR due to an increase in disordered spin on the surface of the NP.^[118] Finally, surface modification can influence the performance of magnetic NPs in MHT. The Brownian relaxation of NPs can be affected by changes in surface coatings which in turn effects the SAR value. Furthermore, the surface coating can act as a ‘bridge’ in heat transfer to the surrounding environment. It has been shown that decreasing PEG chain length can significantly improve the SAR.^[119]

The size and surface properties of NPs dictate pharmacokinetics and biodistribution in vivo in MHT and PTT applications.^[120] NPs between the size of 10 and 100 nm are able to avoid significant uptake by the kidneys (<10 nm) and liver (>100 nm) which increases circulation time. Furthermore, passivation of the NP surface with polymer coatings can also significantly increase circulation time while reducing NP toxicity and improve tumor targeting.^[121,122] Upon in vivo administration of NPs, a protein corona is formed on the surface which ultimately determines the NP fate through changes to the NP size and surface properties.^[123,124] The NP surface properties determine the composition of the protein corona which in turn dictates the trafficking of the NP in vivo. Continued development of techniques to control protein adsorption by tailoring the NP surface to interact with select endogenous peptides and proteins that improve pharmacokinetics and biodistribution will bring great benefits to nanomedicine applications including NP-based HT.

3.2. Hyperthermia Therapy Induced Immune Response

When cells are exposed to elevated temperatures, there are multiple changes that occur. The alteration of membrane characteristics leads to changes in morphology, intracellular changes in sodium and calcium levels, and membrane potential.^[125–128] However, none of these changes are directly associated with cytotoxicity. The direct cytotoxic effects of hyperthermia are thought to be caused by the denaturing and aggregation of DNA synthesis and repair proteins, which causes cell cycle arrest and cell death. Many applications of HT rely on the ablation of tumor cells by heating to temperatures above the fever range. However, in the last few decades, increased evidence indicates that heating tumors to between 39–43 °C provides antitumor immunity and could be particularly useful in combination therapy including as an adjuvant to clinical immunotherapy. ^{[16,129–131]}

3.2.1. Mild Hyperthermia Therapy

Different mechanisms occur at various temperatures and time of exposure which is referred to as the thermal dose. There are several recognized mechanisms for the activation of the immune response by mild hyperthermia. 1) Tumor cells modulate expression of surface markers and other factors when exposed to heat through various mechanisms that can promote an immunogenic response. 2) Heat causes the tumor to express heat shock proteins (HSPs) which activates the host immune response. 3) Heat causes an increase in the release of exosomes containing tumor antigens which in turn activates the host immune response. 4) Heat can directly activate immune cells. 5) Tumor vasculature becomes more permeable at elevated temperatures leading to increased tumor profusion and migration of immune cells to the tumor.^[88]

The modulation of tumor cell surface protein expression can increase their visibility to the immune system. Repasky et al. have shown that cells heated in vitro (39.5 °C, 6 h) are more sensitive to NK cell lysis through increased expression of major histocompatibility complex class I chain-related protein A (MICA), and NKG2D ligand.^[132] Research has also shown that heated tumor cells (43 °C, 30 min) can exhibit increased MHC expression, which, despite the potential for reducing NK cytotoxicity, allows for better recognition by CD8⁺ T cells.^[133] Lysis of tumor cells by NK and CD8⁺ T cells can further improve antitumor immune response by providing a cytokine-driven inflammatory microenvironment.

HSPs are significant participants in immune reactions and the role of HSPs have been one of the most extensively investigated aspects of HT-induced antitumor immune response. HSPs are a heterogeneous family of molecular chaperones that serve a range of functions and are upregulated in cells that are stressed, including through heat exposure.^[134] Of the HSPs, Hsp70 is considered to play the most significant role as an immunostimulant. NK cells recognize an epitope of Hsp70 which stimulates NK cell proliferation and increases cytotoxic activity.^[135–137] When released from tumor cells, Hsp70 binds directly to APCs, including DCs, and activates cytokine production and antigen uptake.^[138–141] In the extracellular environment, Hsp70 is often associated with tumor antigens and can transfer these antigens to APCs. The APCs are then able to elicit a CD8⁺ T cell response through cross-presentation via MHC class I.

Despite the immunostimulatory role of Hsp70 and many other HSPs, there is considerable evidence that some HSPs inhibit antitumor immune response. Hsp90, for instance, interacts with the tumor suppresser protein p53, and subsequently inhibits apoptosis.^[142] Another example is Hsp110, which despite its ability to stimulate DCs to produce inflammatory cytokines and prime naïve T cells, can also reduce DCs’ antitumor immune activities when bound to scavenger receptor A. These confounding results indicate that further research on the role of various thermal doses and their effects on the expression levels of the various HSPs must be carried out to determine optimal antitumor treatment conditions.

Exosomes, which are small membrane vesicles, are released by cells and play a role in intracellular communications. Tumor-derived exosomes are enriched with tumor antigens and may serve as a potential immunostimulatory factor.^[143] Tumor cells exposed to stress, including heat, have shown increased release of exosomes.^[144] Studies show that DCs exposed to tumor-derived exosomes are able to in turn display those antigens and activate a tumor antigen-specific CD8⁺ T cell response.^[145] Cao et al. have shown that exosomes from heated tumors increase the antitumor immune response. In these studies, the tumor-derived exosomes were shown to act as an antigen source for APCs. DC activation led to tumor-specific CD8⁺ T cell response in a transgenic mouse model.^[146,147] Furthermore, chemokines contained within the exosomes where shown to attract DCs, and CD4⁺ and CD8⁺ T cells. While not reported in the context of heat stress, there is research that indicates tumor-derived exosomes can generate immunosuppressive activity. Some tumor-derived exosomes have shown to contain ligands such as FasL and TRAIL, which trigger apoptosis of activated T cells.^[148] Exosomes can also contain NKG2D ligands which block NKG2D receptors and suppress NK cell and CD8⁺ T cell cytotoxicity.^[149] Additionally, some tumor-derived exosomes aid in the differentiation and function of suppressor cells and Tregs. Further research is needed to determine if these immune suppressive mechanisms are stimulated by the heating of tumors.

Direct heating at fever levels has been shown to directly stimulate immune cells in vitro. The heating of CD8⁺ T cells (39.5 °C, 6 h) in vitro increased production of antigen-specific IFN-γ and improved tumor cell killing.^[150] Culture of bone marrow derived DCs in vitro at elevated temperatures (39.5–41°C, 6–24 h) has shown to activate DCs by upregulation of MHC class I and II, CD40, CD80 and CD86. Furthermore, the activated DCs demonstrated improved induction of T cell proliferation.^[151,152] IL-2 activated NK cells have been shown to display improved lysis activity in vitro after heating (39 °C, 6 h), which was associated with clustering of NKG2D surface receptors.^[132] It has also been reported that macrophages cultured at elevated temperatures (39.5–40 °C, 2–3 h) have demonstrated polarization to the pro-inflammatory state.^[153–155] The majority of the literature indicates these direct effects of hyperthermia on immune cells predominantly takes place in the fever range, but not at higher temperatures.

Abnormal tumor vasculature presents one of the major challenges in cancer therapy by inhibiting delivery of therapeutics. The use of HT has shown to increase the diameter of tumor vasculature resulting in improved perfusion at temperatures below 43 °C.^[156] This increased perfusion has shown to improve oxygenation levels and increase the delivery of therapeutics.^[157,158] Importantly, increased tumor profusion may facilitate the trafficking of immune cells within the tumor space, further improving the antitumor immune response of HT.^[159]

3.2.2. Thermal Ablation Hyperthermia Therapy

The use of HT at higher temperatures to ablate tumors, a common practice in PTT applications, primarily causes necrosis which in turn induces an immunogenic cell death.^[160,161] The desired temperature for ablative application is >50°C to ensure significant tumor destruction or complete annihilation.[16] Under severe heat shock conditions typically associated with tumor ablation, calreticulin which serves as an “eat me” signals is upregulated on the surface of tumor cells.^[162] There is evidence to suggest increased reactive oxygen species (ROS) production mediated by HT leads to the increase in calreticulin expression.^[163–165] In addition to calreticulin upregulation under ablative conditions, danger-associated molecular patterns (DAMPs) including HSPs, S100 proteins, and high-mobility group box 1 are exposed on the cell surface or released into the extracellular space.^[166] DAMPs facilitate the engulfment of dying tumor cells by immune cells, stimulate antigen presentation, and primes an anti-tumor T cell response.^[162] A drawback of tumor ablation is elevated temperatures in adjacent healthy tissue which can cause off target toxicity.

3.2.3. NP-mediated Subcellular Hyperthermia Therapy

The ability to target NPs to subcellular structures potentially allows for destruction of subcellular targets with highly localized heating that does not cause a macroscopic temperature rise. ^[93] Localized heating using EGFR-targeted NPs has shown to be effective in killing cancer cells where the overall sample temperature remained at 37°C.^[167] It has been speculated that the possible mechanism behind this increased cell death was the thermal/mechanical actuation of the EGFR apoptotic pathway. In a separate study, EGFR-targeted NPs were shown to accumulate in lysosomes, which led to disruption of the lysosomes and increased cell toxicity.^[168] Additionally, mitochondria-targeted NPs have demonstrated the ability to enhance apoptosis of cancer cells with only subcellular localized heating.^[169] It is well accepted that iron oxide NPs can serve as a Fenton reagent and produces ROS. Under AMF stimulation, the ROS production of iron oxide NPs is enhanced and can lead to tumor cell apoptosis.^[170] Magnetic NPs targeted to lysosome have shown to cause cell death by local temperature increase at the periphery of the NP which enhanced ROS production through the Fenton reaction.^[171] The effect of localized heating and ROS production induced lipid peroxidation, lysosomal membrane permeabilization, and leakage of Cathepsin-B which led to cancer cell death. While the application of hyperlocal heating is promising, continued research is needed to elucidate the mechanisms behind the efficacy associated with subcellular targeting and heating of specific organelles and cellular structures.

4. Combined Nanoparticle-Based Hyperthermia and Immunotherapy

Combination therapy, where treatment combines two or more therapeutic agents, has become a significant focus in clinical cancer therapy. Combination therapies are capable of providing synergistic benefits that can maximize efficacy and reduce acquired resistance to treatment through the targeting of multiple cancer pathways. Augmentation of chemo- and radiotherapy with HT has gained significant clinical interest as evidenced by multiple phase II and III trials.^[172,173] However, HT as an augmentation therapy to immunotherapy has not yet been assessed in clinical studies, but significant preclinical evidence strongly supports clinical investigation.^[174]

The diverse mechanisms of HT immune stimulation, demonstrated through in vitro and some in vivo studies, may provide a wide range of potential strategies to improve clinical outcomes. HT can serve to shorten treatment times, allow for protracted and repeated use, and potentiate multifactorial immune effects while maintaining a favorable toxicity profile. While considerable research needs to be undertaken to elucidate the required HT thermal dose to induce the immune responses desired for a given immunotherapy treatment, there is some evidence that minimal heating times may be sufficient for many applications. Shorter treatment times would provide more favorable treatment schedules and may facilitate more optimized treatment timelines with immunotherapy. Currently, checkpoint inhibitor therapies including anti-PD-1 and anti-CTLA 4 therapy used for front-line treatment of metastatic melanoma and lung cancer require extended course therapy. Patients typically receive treatment every 2–3 weeks over the course of several months or even years. Importantly, there are no limitations to the repeated use of HT as opposed to chemo- and radiotherapy. HT may provide alternatives to clinically relevant combined immunotherapies. For example, combined anti-PD-1 and anti-CTLA 4 therapy has shown great potential in providing relapse-free survival for the treatment of metastatic melanoma, but is hindered by significant toxicity.^[175] Response to checkpoint inhibitors is highly correlated to the number of CD8⁺ and CD4⁺ T cells within the TME. HT has shown to increase the number of these important immune cells and may prove to be a safer alternative to more toxic combined immunotherapies.

For HT to reach its clinical potential as an immunotherapy adjuvant, several limitations need to be addressed: these include 1) the shallow depth of penetration for some HT techniques which limits application to superficial tumors, 2) the inability to adequately focus energy for techniques capable of reaching deeper tissue, which heats regions beyond the target tissue, and 3) the inability to provide even, controlled heating to ensure the proper thermal dose is delivered for a given application. The incorporation of NPs in HT addresses these limitations by providing a noninvasive platform with some formulations capable of reaching deep tissue tumors while providing tumor specificity through passive, size-based accumulation or molecularly targeted approaches.^[176] Additionally, NPs can reduce temperature heterogeneity to elicit a more even response across the tumor for a given thermal dose.^[177,178] Intravenously delivered NPs of appropriate hydrodynamic size (~10–100 nm), preferentially accumulate within tumors due to the EPR effect which is caused by rapid vasculature growth in tumors that leads to leaky vasculature with poor lymphatic drainage.^[120,179] Additionally, the flexible surface chemistry and large surface-area to volume ratios of NPs provides the ability to functionalize NPs with targeting agents such as peptides and antibodies targeted to overexpressed tumor receptors. A potential limitation of NP-based HT has been insufficient tissue penetration. Several methods have been employed to improve tissue penetration including the use of NPs that are able to reduce size through environmentally induced degradation and through functionalization of NP surfaces with factors that are able to restructure the extracellular matrix.^[180] However, preclinical evidence suggests HT can induce a broad tumor-specific immune response by heating only a portion of the tumor, which may lower the bar for successful treatment in combination with immunotherapy.

Once accumulated in tumors, NPs can be activated non-invasively to facilitate HT with external sources of light, magnetic fields, or radiofrequency radiation.^[181–183] NPs can facilitate HT-based combination therapies by coadministration of NP and therapeutics, or through incorporation of therapeutics directly into the design of the NP. These advantages provide the potential to facilitate novel combinatorial approaches such as NP-mediated gene silencing of immune check point ligands and mild NP-based HT to facilitate a synergistic response (Figure 2). In addition to providing a non-invasive heating source, many MHT and PTT NPs in HT applications can facilitate real-time imaging using MR, CT, photoacoustic, and/or X-ray imaging to aid in development and optimization of treatment time courses.^[179]

Figure 2.

The synergistic effects of mild NP-based HT and immunotherapy in tumors. a) Systemically administered NPs localize to the primary tumor and metastatic cells. b) The NPs release siRNA against PD-L1 (siPD-L1), which inhibits translation of PD-L1 via the RNA-silencing complex (RISC). Irradiation with an external AMF or NIR light initiates an HT induced immune response that increases the release of exosomes containing TSAs, and expression of HSPs and MHC receptors. Exosome released TSAs and HSP-TSA complexes activate APCs which release NK cell activating IFN-α, and are trafficked to the lymph nodes where they activate T cells. c) Activated T cells traffic to the primary and metastatic tumors, initiating TCR mediated killing of tumor cells. Gene silencing by siPD-L1 creates a positive feedback loop which allows an uninhibited immune response, resulting in primary tumor cell cytotoxicity, abscopal effects, and immune memory.

PTT and MHT are similar in that they both provide localized NP-based heating, however, there are significant differences between the two platforms. First, MHT and PTT require different external energy sources. Furthermore, due to the use of light as the external energy source, PTT is limited to shallow penetration depths while MHT has virtually no depth limitations. It is generally recognized that MHT’s main mode of efficacy in cancer therapy is HT mediated apoptosis driven by several factors including an increase in Hsp70 and ROS production which can induce an immune response.^[164,165] PTT has typically been utilized for ablative treatment that leads to necrosis and immunogenic cell death. However, PTT can also be used for mild HT and recent research has shown that mild HT application of PTT can be more effective in combination with immunotherapy than ablative approaches.^[129]

Multifunctional NPs can provide means to augment NP-based HT with additional therapeutic approaches utilizing a single probe. For example, researchers have utilized antiferromagnetic pyrite polyethylene glycol nanocubes to provide simultaneous chemodynamic therapy and PTT.^[184] CuS based NPs have been used to generate a multifunctional NP that facilitates radiotherapy and PTT as a synergistic combination therapy.^[185] In another approach, a nanohybrid with Cu contained in layered double hydroxide facilitates PTT while FeOOH nanodots provide ROS production that amplified the immune response.^[186,187] Graphene oxide-grafted magnetic nanorings have shown the ability to enhance ROS production at mild temperatures (40°C) in a hypoxic tumor environment to induce calreticulin surface expression and produce a robust immune response including polarization of macrophages to the M1 state and an increase in infiltrating T lymphocytes.^[188] Integrating NPs with these multifunctional therapeutic capabilities with existing clinical cancer therapies such as immunotherapy, may open new avenues for treatment.

5. Applications in Nanoparticle-Based Photothermal Therapy and Magnetic Hyperthermia Therapy Combined with Immunotherapy

NP-based HT is a promising adjuvant to immunotherapy because of its ability to modulate immune responses by facilitating activation of immune cells and increasing tumor permeability. The potential synergistic effects of combined NP-based HT and immunotherapy may address the limitations of current immunotherapies and broaden their application in the clinic. This section will highlight recent progress in the application of NP-based HT to address limitations of clinically relevant cell-based, and ligand/receptor based immunotherapies, including ICB.

5.1. Cell-Based Therapies

Cell-based immunotherapies, including adoptive T cell therapy, were some of the earliest forms of clinical immunotherapy and remain a key focus in development of new therapeutic approaches. Research in the incorporation of NPs into various aspects of cell-based immunotherapies has gained significant attention over the last two decades.^[189–191] NPs have been used to label and provide real-time tracking of immune cells in basic immunology research and to track cell movement in antitumor immunotherapies that are under development. Furthermore, the role of functionalized NPs as an immunostimulant, including via HT, is an area of increasing interest in immunotherapy.^[191,192] The ability of NP-based HT to modulate APC and effector cell functions, provides significant opportunity to augment existing cell-based immunotherapies.

The application of MHT in combination with immature DC injections was first evaluated by Tanaka et al.^[193,194] In these studies, magnetic cationic liposomes formed by sonication of a mixture of magnetite particles and cationic liposomes were used to provide localized heating to B16 melanoma tumors in a syngeneic mouse model. Immature DCs were injected 1 day after HT. Complete tumor regression was observed in 60% of the mice in the group receiving HT and dendritic cell injections, while no tumor regression was observed in mice receiving only HT or DC therapy. An increase in cytotoxic T lymphocyte and NK cell activity was observed in ex vivo assays using splenocytes from the cured animals. Importantly, cured mice rejected a secondary challenge of B16 melanoma cells indicating that NP-based MHT combined with DC therapy led to prolonged tumor immunity.

NPs can be used as carrier platforms for encapsulation of, or surface modification with TAAs or immunomodulating molecules for delivery to APCs.^[195,196] These approaches have been shown to propagate T cell expansion, activation, and response.^[196] Recent research by Zhang et al., has incorporated this idea with NP-based PTT.^[197] Gold NPs (AuNPs) were generated cellularly by incubation of B16F10 murine melanoma cells with HAuCl₄, then removed through exocytosis of vesicles containing AuNPs. AuNPs prepared in B16F10 cells were then introduced into DCs to promote the antigen presenting capabilities of AuNPs. After incubation in DCs, UV irradiation was used to secrete the AuNPs. The NPs were ~40 nm with a core-shell structure of ~6–8 nm indicating significant protein corona formation. Proteomic studies showed the protein corona of AuNPs synthesized in melanoma cells and incubated in DCs had 249 proteins intrinsic to the DCs and 56 associated with B16F10 melanoma. When used as a monotherapy in a syngeneic mouse model the AuNPs showed a modest inhibitory rate (61.6%) indicating potential antitumor immunity from the antigen presenting NP alone. However, when NIR irradiation was incorporated to facilitate HT, a tumor inhibitory rate of 96.7% was observed along with anti-metastatic behavior and long-term survival. The likely mechanism behind the improved tumor response was the synergistic effect of both antigen presenting AuNPs and HT. Antigen presenting AuNPs alone showed a modest increase in CD4⁺ and CD8⁺ T cell proliferation and activation while tumors receiving both AuNPs and NIR irradiation showed significant promotion of proliferation and activation. A major implication of this work is the potential for NPs to facilitate personalized therapy through incorporation of patient-specific TAAs on the NP surface.

CART therapy represents one of the more promising approaches in current clinical immunotherapy. CART therapy to date has focused primarily on hematological cancers, yet holds great potential in treatment of solid tumors because they can be engineered to target virtually any tumor. However, solid tumor present several challenges not found in hematological cancers. Identification of appropriate antigens has been a challenge, but perhaps more significantly, is the inefficiency of CAR T cell trafficking in solid tumors and the T cell suppressive, pro-tumor TME. Recent work by Chen et al. aimed to address these challenges using poly(lactic-co-glycolic) acid (PLGA)-encapsulated indocyanine green (ICG) NPs (PLGA-ICG) (Figure 3).^[198] ICG is a NIR dye that allows for PTT. WM115 melanoma cells were inoculated in both flanks of Nod scid gamma mice. PLGA-ICG was injected intratumorally on one flank followed by irradiation. Two hours post PTT, CAR T cells targeting the antigen chondroitin sulfate proteoglycan-4 (CAR.CSPG4⁺), which is overexpressed in melanoma and glioblastoma, were administered intravenously. Post PTT, tumor vasculature morphology appeared dilated and the interstitial fluid pressure (IFP) reduced compared to control tumors. An increase in tumor profusion was further corroborated by a decrease in hypoxia-inducible factor (HIF)-1α which indicates increased oxygenation. Importantly, biodistribution studies using IVIS imaging, flow cytometry, and immunofluorescence imaging showed increased localization of CAR.CSPG4⁺ T cells in the PTT treated tumor. Furthermore, tumor growth was significantly suppressed at day 20 post-treatment with 2 out of 6 mice macroscopically tumor free when treated with combined PTT and CAR.CSPG4⁺ T cells. Measured cytokine levels showed an increase in pro-inflammatory IL-6 for PTT treated mice and IL-2 and IFN-γ released from CAR T cells was increased in the combined therapy. These results indicate the multifaceted way NP-based HT can positively augment existing immunotherapies and open new avenues to adoptive T cell therapies in a wide range of tumor types.

Figure 3.

Combined photothermal ablation and adoptive transfer of CAR T cells promotes T cell infiltration, mediates tumor hypoxia, and inhibits the growth of the human melanoma. a) Schematic illustration showing the effects of the mild heating of the tumor that causes enhanced infiltration and activation of adoptive transfer CAR.CSPG4⁺ T cells. b) In vivo Bioluminescence quantification of CAR.CSPG4⁺ T cells detected in the tumor with or without photothermal ablation. Data are presented as mean ± s.e.m. (n = 3). c) Representative hypoxia and HIF1-α immunofluorescence staining of the tumors after photothermal therapy (Scale bar: 50 μm). d) Average tumor growth kinetics in different groups. Day 0 indicates the day in which treatment was initiated. Data are presented as mean ± s.e.m. (n = 6). e) Murine IL-6 levels detected in the tumors 7 days after the indicated treatments. Data are presented as mean ± s.e.m. (n = 8). f,g) Human IL-2 and IFN-γ levels detected in the tumor 7 days after the indicated treatments. Data are presented as mean ± s.e.m. (n = 7). Statistical significance was calculated via one-way ANOVA with a Tukey post hoc test. P value: *P < 0.05, **P < 0.01, and ***P < 0.001. Adapted with permission.^[198] 2019, Wiley.

5.2. Ligand and Receptor-Based Therapies

5.2.1. Immune Regulator IL-2

ILs are cytokines that mediate the crosstalk between an array of immune cells. IL-2 regulates the differentiation and growth of T cells and some B cells and has long been of interest as a cancer immunotherapy.^[199,200] IL-2 has showed potential in treating metastatic cancers, yet its clinical applications remain relatively restricted due to several shortcomings. First, IL-2 plays a dual role with T cells with its ability to increase expansion of both effector T cells and Tregs, which some studies have used to enhance antitumor immune response and others have used to reduce immune response. Furthermore, IL-2 has exhibited severe toxicity due to its short serum half-life, which requires it to be given at a high dose.^[200] Despite these obstacles, IL-2 therapy may benefit from the immunostimulatory effects of HT. An early study evaluating the feasibility of NP-based MHT using iron oxide-based magnetic liposomes demonstrated the potential of IL-2 therapy combine with HT in a B16 mouse melanoma model.^[133] In vitro results showed the HT increased Hsp70 expression and increased the infiltration of CD8⁺ T cells and Mac-3-positive macrophages/DCs. In vivo studies showed that combined HT and IL-2 therapy led to complete regression in 75% (6 of 8) mice inoculated with B16 melanoma. A more recent study using iron oxide NPs produced similar results in a Lewis lung carcinoma mouse model.^[201] In vivo immunohistochemical analysis showed that combined therapy led to increased expression of Hsp70, and infiltration of both CD4⁺ and CD8⁺ T cells. While this study did not evaluate overall survival, the tumor growth inhibitory rate for combined therapy was considerably higher (68.1%) as compared to IL-2 (14.2%) or MHT (45.8%) alone. These results indicate IL-2 therapy may be enhanced by the immunostimulatory effects of HT, however further studies are needed to better determine the role of HT in augmenting Tregs expansion known to be caused by IL-2 and the potential of HT to reduce the required IL-2 dose, thus addressing the toxicity associated with high dose IL-2 therapies.

5.2.2. Small Molecule Agonists

Several small molecule agonists have demonstrated potential as a cancer immunotherapy by targeting various pathways that lead to the immunosuppressive TME. Several of these agonists targeting Toll-like receptors (TLRs) 7, 8, and 9 have recently been used in conjunction with NP-based HT. TLRs play a fundamental role in bridging innate and adaptive immunity.^[202] Activated TLRs induce a pro-inflammatory response, and are fundamental in the development of antigen specific immunity.^[203] TLRs can be triggered by single stranded RNA during viral infections, but can also be triggered by agonists that share similar structures to nucleosides. When triggered, TLRs exhibit a powerful immune stimulatory action which has significant potential as a therapeutic approach. One prominently researched TLR-9 agonist is the synthetic oligodeoxynucleotide containing unmethylated CG dinucleotides (CpG).^[204] CpG demonstrated efficacy in several preclinical studies, yet clinical trials yielded limited success. CpG was well tolerated and showed promising effects such as an increase in pro-inflammatory cytokine release, however, other positive effects including elevated number of NK cells and CD8⁺ T cells were only occasionally observed and mostly on the periphery of tumors.^[205]

Recently, research has indicated that CpG may still play an important role in immunotherapy when combined with other therapeutic approaches.^[206] Li et al. utilized a gold-based photothermal CpG nanotherapeutic (PCN) to induce fever range heat to augment CpG immunotherapy.^[207] Tumor growth was significantly suppressed when mild heat was added as compared to CpG alone in a 4T1 murine breast cancer model. Notably tumors in 4 out of the 5 mice receiving PCN and light to induce heat disappeared. The addition of fever-range heat enhanced a range of effects, including enhancement of tumor profusion, promotion of DC maturation, and an increase in tumor-infiltrating CD45⁺ leukocytes, positively affecting CpG delivery and efficacy. Similar results were obtained in two PTT-based systems that highlight the utility of NP-based approaches. Wu et al. utilized a graphene quantum dot loaded with fluorophore-labeled CpG and functionalized with a photosensitizer to facilitate PTT and photodynamic therapy, and Gd³⁺ to facilitate MR imaging.^[208] This multifunctional NP enhanced CpG therapy and provided bimodal MR/fluorescence imaging in vivo. Guo et al. utilized a magnetic-responsive immunostimulatory nanoagent (MINP) to facilitate magnetically targeted CpG delivery combined with PTT in a 4T1 murine breast cancer model (Figure 4).^[209] Superparamagnetic iron oxide served as the photosensitizer, magnetic targeting agent, and served as a contrast agent in MR and photoacoustic imaging to track the magnetically targeted delivery of CgP to 4T1 tumors. Increased DC maturation, IL-6 and IFN-α levels, and T cell infiltration were observed in tumors receiving magnetic targeting and PTT. Near total tumor suppression was observed in mice receiving MINP combined with magnetic targeting and PTT. Small molecular agonist against TLRs 7 and 8 in combination with PTT has also shown promise. Chen et al. utilized the TLR7/8 agonist, R848, and the photosensitizer polyaniline grafted on to glycol chitosan to form a self-assembled NP (R848@NP).^[129] R848@NP in combination with NIR both inhibited tumor growth and provided long-term immunity in rechallenged mice.

Figure 4.

In vivo immunostimulatory and anti-tumor effects of MINPS-based PTT. a) Schematic illustration of the synthetic process for the MINPs (CpG@PLGA-PLL-mPEG/SPIO). b) DC maturation levels in the primary tumor (1st) cells collected on day 3 after the aforementioned treatments by flow cytometry. *p<0.05, **p<0.001: MINPs + magnetic field (MF) + Laser compared to control groups. c) The secretion levels of TNF-α and IL-6 by ELISA assay in the sera from the mice on day 3 after the aforementioned treatments on the primary tumors (1st), respectively. *p<0.05, **p<0.001: TNF-α level in group of MINPs + MF + Laser compared to control group. #p<0.05, ##p<0.001: IL-6 level in group of MINPs + MF + Laser compared to control group. d) Immunofluorescence images of effector CD8+ T cells and IFN-γ in the distant tumors (2nd) on day 7 after different treatments on the primary tumors (1st). The tumor specimens were sectioned and immunostained with anti-CD8-FITC (green) and anti-IFN-γ-Cy3 (red), respectively. e) Growth curves of the primary tumors (1st) and f) the distant tumors (2nd) on mice in different groups after the aforementioned treatments. **p<0.001 compared to control group. L = NIR light. Adapted with permission.^[209] 2019, Elsevier.

5.2.3. Immune Checkpoints

Despite the success of ICB immunotherapies in hematological cancers, metastatic melanoma, non-small cell lung cancer, as well as several other types of cancer, widespread use in the clinic has not been realized. Even in the most responsive cancers, many patients do not experience clinical benefits and survival gains are modest in most cancers. Recent research has shown that the TME plays a significant role in the response of cancers to ICB. The concept of “hot” versus “cold” tumors has gained acceptance among immuno-oncologists, where these terms are defined as tumors containing more than a defined threshold of inflammatory cells (hot), or tumors that do not (cold).^[210] This dichotomy is thought to be largely responsible for the heterogeneity in response to ICBs where “cold” tumors do not respond as well as “hot” tumors. The TME of many cancers, in particular solid tumors, tend to fall into the category of “cold”. HT is a promising adjuvant to immunotherapy because it has the ability to turn “cold” tumors “hot” by increasing the proinflammatory response within the tumor, reducing hypoxia that leads to significant immune suppression, and increasing perfusion allowing immune effector cells to reach deep within tumors. There has been significant pre-clinical progress made in the application of NP-based HT to address the limitations in ICB therapy with the goal of broadening the application of ICB in the clinical setting.

In an effort to increase efficacy in ICB targeting the PD-1 pathway, several groups have developed NP-based HT systems to augment anti-PD-1/PD-L1 therapy. Zhang et al. have developed an NP-based PTT platform, capable of providing PTT and simultaneously deliver anti-PD-1.^[211] The NP is comprised an outer layer of PLGA which is functionalized with PEG, iron oxide NPs, and a tumor targeting peptide (GRGDS) (Figure 5). Within the PLGA layer resides a solution of perfluoropentane (PFP) containing anti-PD-1. Upon laser excitation, the PFP is evaporated through a process termed optical droplet vaporization, which in turn degrades the NP, releasing anti-PD-1. The iron oxide NP on the surface of the PLGA layer serves to increase energy absorption, thus facilitating PTT. In vivo studies showed the NP was able to increase pro-inflammatory cytokine levels. Efficacy studies in a B16F10 murine melanoma model showed almost complete inhibition of tumor growth and a significant increase in survival when anti-PD-1 was delivered by the NP and combined with PTT, whereas, free anti-PD-1 combined with PTT demonstrated modest tumor inhibition (43.2%). Control tumors and free anti-PD-1 treated tumors showed limited T cell infiltration while those treated with anti-PD-1 NPs and PTT exhibited significant T cell infiltration, indicating ability to transform the tumor from “cold” to “hot”. These results were obtained using intravenous (i.v.) injection of NP which is significant because this route of administration is less invasive than direct tumor injections which is often relied on when active targeting strategies are not incorporated into the NP design to facilitate high uptake in tumors. Liu et al. utilized a star-shaped gold NP (GNS) with strong plasmonic properties due to tip enhancement plasmonics that amplifies laser light to facilitate i.v. injections while demonstrating antitumor activity in a glioblastoma murine model.^[212] Importantly, GNS PTT combined with anti-PD-L1 demonstrated greater tumor growth inhibition than treatment with anti-PD-L1 alone. Tumor-free survivors from the initial efficacy study were rechallenged in the contralateral flank using CT-2A tumors with the majority of mice rejecting the rechallenge. Ge et al., utilized iron oxide NPs for PTT in combination with anti-PD-1 therapy. In this work, the magnetic properties of the iron oxide NP allowed for magnetic targeting to achieve increased uptake of i.v.-administered NPs in 4T1 murine breast cancer.^[213] PTT in combination with anti-PD-1 facilitated DC maturation, eradicated tumors, and prevented lung metastasis which often occurs in the 4T1 model. The prevalence of CD8⁺ T cells, B cells, and NK cells was evaluated in a distant tumor (opposite flank, untreated) and showed a significant increase in the number of these cells when anti-PD-1 was combined with PTT as compared to anti-PD-1 alone. Other groups have taken various approaches to increase magnetic susceptibility or photothermal capabilities to improve magnetic targeting and/or the efficiency of AMF/light-to-heat conversion. Wang et al. developed a highly efficient PTT NP synthesized from copper sulfide which was also capable of antigen capturing.^[214] To improve magnetic susceptibility of NP utilized in MHT in combination with ani-PD-1 therapy, researchers have anchored iron oxide NPs in titanium sulfide nanosheets^[215], doped iron oxide NPs with cobalt^[216], and produced highly magnetic ring structured iron oxide NPs.^[217]

Figure 5.

GOP@aPD1-based PTT delivers aPD1 to tumors and sensitizes tumors to PD-1 blockade immunotherapy for the treatment of melanoma. a) The compositions of GOP@aPD1 NPs (left). After laser irradiation, GOP@aPD1 NPs dissociate, releasing aPD1 and iron oxide nanoparticles (right). (b-d) Cytokine levels in sera from mice isolated at 24, 72 and 168 h after different treatments (control, PTT and GOP@aPD1- based PTT). The data are shown as the mean ± SD, n=3 per group. e) The relative tumor growth curves during the various treatments over the 14-day study period (mean ± SD, n=5, **p < .01). f) Survival curves of the treated and control mice (*p < .05). g) Representative CLSM images of the residual tumors in G1 (upper) or G8 (median; under) showed CD3+ T cell, CD4+ T cell and CD8+ T cell infiltration after immunofluorescence staining. G1 = Saline control, G2 = Free anti-PD-1, G3 = PTT only, G4 = Free anti-PD-1 + PTT, G5 = iron oxide loaded NP + PTT, G6 = anti-PE-1 loaded NP + PTT, G7 = iron oxide and anti-PD-1 loaded NP, G8 = iron oxide and anti-PD-1 loaded NP + PTT. Adapted with permission.^[173] 2019, Elsevier.

Preclinical studies have also looked at ICB based on anti-CTLA-4 antibodies combined with NP-based HT systems. Chao et al. developed an MHT NP composed of pure iron (FeNP) which was highly efficient in thermal conversion. The increased magnetic susceptibility allowed for efficient magnetic targeting of i.v.-administered NPs. MHT was utilized in combination with anti-CTLA-4 to treat flank 4T1 tumors in mice. Tumors treated with the combination therapy were completely ablated and animals survived for more than 60 days. Abscopal effects were demonstrated using a T26 colon cancer model and long-term immune memory was demonstrated in rechallenge experiments. The results of this study were confirmed in two orthotopic models using B16 melanoma and 4T1 breast cancer cells. Chen et al. showed that NP-based PTT combined with anti-CTLA-4 was able to deplete Tregs and increase CD8⁺ T cell infiltration in a 4T1 murine model demonstrating the ability of NP-based HT to produce an immunogenic TME in combination with ICB.

6. Conclusions and Outlook

In recent preclinical research, NP-based HT has shown potential to serve as a powerful treatment platform in combination with immunotherapy. The application of magnetic targeting approaches and functionalization of NPs with targeting agents have improved tumor-specific heating, while development of new NP formulations and morphologies have led to increased efficiency in thermal conversion. Together, these advancements begin to address limited tumor uptake of NPs which has been one of the greatest hurdles to the broad application of NP-based HT. NP-based HT has demonstrated in preclinical studies the ability to increase tumor immunogenicity through a range of immune system mechanisms and increase permeability of solid tumors, addressing two major factors limiting broader application of immunotherapy in the clinic. Furthermore, the multifunctionality of NP architecture provides the potential to combine HT and immunotherapy delivery in a single probe and facilitate administration of other treatment modalities including chemo- and radiotherapy in parallel to HT.

Despite recent advancements, further progress is needed for NP-based HT to achieve clinical success as an adjuvant to immunotherapy. Research in the development of NPs that better evade clearance organs while exhibiting high affinity for tumor tissue is paramount to address safety and efficacy concerns. Approaches should include NP surface modifications to minimize or control opsonization and mitigate macrophage recognition while constraining size to within 10–100 nm, and incorporation of targeting ligands with high tumor specificity. Furthermore, the safety profiles of new NP formulations tailored for optimal thermal heating efficiency by modification to size, shape, and/or composition must be thoroughly evaluated in vivo to address the potential toxic effects of these formulations.

To provide a strong synergistic response in combined NP-based HT and immunotherapy, a better understanding of the relationship between thermal doses and specific immune responses in NP-based HT applications is needed. While there is significant research elucidating the relationship between thermal dose and the immune response, there is also considerable disagreement in the literature. To address this issue, improvements in temperature monitoring techniques combined with a systematic evaluations of the thermal dose response of NP-based HT needs to be undertaken. Furthermore, the potential effects of immunotherapy on the immune response to HT in combined approaches should be investigated to better understand the synergy between these therapies. Answering these challenges may open the door for broad application of NP-based HT in combination with immunotherapy in the clinic.

Biographies

Zachary Stephen is a research associate in the Department of Materials Science and Engineering at the University of Washington in Seattle, WA. His research focuses on the development and application of nanomaterials in cancer therapy with an emphasis on development of iron oxide nanoparticles for targeted delivery of chemo and gene therapies.

Miqin Zhang is Kyocera Chair Professor of Materials Science & Engineering and Neurological Surgery, and adjunct professor in the Departments of Bioengineering, Radiology, and Orthopaedics & Sports Medicine at the University of Washington (UW). She received her Ph.D. from University of California at Berkley. Her research focuses on nanomedicine for cancer diagnosis and treatment, biomaterials for tissue engineering, and biosensors for detection of chemical and biological agents.