Harnessing Hyperthermia: Molecular, Cellular, and Immunological Insights for Enhanced Anticancer Therapies

Abstract

Hyperthermia, the raising of tumor temperature (≥39°C), holds great promise as an adjuvant treatment for cancer therapy. This review focuses on 2 key aspects of hyperthermia: its molecular and cellular effects and its impact on the immune system. Hyperthermia has profound effects on critical biological processes. Increased temperatures inhibit DNA repair enzymes, making cancer cells more sensitive to chemotherapy and radiation. Elevated temperatures also induce cell cycle arrest and trigger apoptotic pathways. Furthermore, hyperthermia modifies the expression of heat shock proteins, which play vital roles in cancer therapy, including enhancing immune responses. Hyperthermic treatments also have a significant impact on the body’s immune response against tumors, potentially improving the efficacy of immune checkpoint inhibitors. Mild systemic hyperthermia (39°C-41°C) mimics fever, activating immune cells and raising metabolic rates. Intense heat above 50°C can release tumor antigens, enhancing immune reactions. Using photothermal nanoparticles for targeted heating and drug delivery can also modulate the immune response. Hyperthermia emerges as a cost-effective and well-tolerated adjuvant therapy when integrated with immunotherapy. This comprehensive review serves as a valuable resource for the selection of patient-specific treatments and the guidance of future experimental studies.

Hyperthermia in Cancer Treatment

Hyperthermia in cancer treatment has the potential to enhance the effectiveness of radiotherapy, chemotherapy, and immunotherapy ¹ while maintaining low toxicity. ² This enhancement is achieved through multiple mechanisms, including the promotion of increased blood flow within the tumor microenvironment (TME), facilitating lymphocyte infiltration and oxygenation, ³ and optimizing drug penetration. ⁴ Furthermore, heat disrupts DNA repair enzymes, which are often involved in therapy resistance. ⁵ Heat shock triggers apoptosis or necrosis in tumor cells, ⁶ leading to altered surface marker expression ⁷ and the release of cellular debris that serves as antigens, thereby initiating an antitumor response. ⁸

Despite its initial appeal as a cost-effective and well-tolerated treatment modality, the integration of hyperthermia into clinical studies has only recently gained consideration. Early trials in the early 1900s using electromagnetic radiation and later, in the 1950s, involving microwaves, yielded varying results that were met with skepticism. ⁹ In theory, what initially appeared as a straightforward treatment option turned out to be challenging to optimize in specific anatomical sites without appropriate heating techniques and temperature monitoring. ¹⁰ The effectiveness of hyperthermic monotherapy in treating superficial tumors was reevaluated in the 1980s, revealing response rates of up to 40%. ¹¹ Although hyperthermic therapy alone can contribute to tumor remission, ¹² it is most effective when used in combination with other treatment modalities including radiotherapy^13,14 and chemotherapy.^2,15,16

In recent years, there has been a significant increase in the number of clinical trials examining hyperthermic treatment, with hyperthermic intraperitoneal chemotherapy (HIPEC) emerging as a widely applied approach. ¹⁷ HIPEC involves the administration of heated chemotherapy fluids into the abdominal cavity and is commonly used in the treatment of gastrointestinal and gynecological cancers. ¹⁸ HIPEC following cytoreductive surgery (CRS) has gained broad acceptance in the treatment of colorectal ¹⁹ and gastric cancers ²⁰ with peritoneal metastases. Repetitive CRS and HIPEC have shown effectiveness in managing appendiceal cancer. ²¹ Additionally, HIPEC may hold promise as a prophylactic therapy to prevent peritoneal recurrence in high-risk individuals. ²²

In the context of stage III ovarian cancer, HIPEC treatment following CRS has demonstrated improved long-term survival compared to surgery alone. ²³ In a multicenter randomized Phase 3 trial, 245 patients with stable disease after 3 cycles of carboplatin and paclitaxel were allocated to undergo interval CRS, either with or without the addition of HIPEC containing cisplatin. The surgery-plus-HIPEC group exhibited a higher median recurrence-free survival of 14.2 months compared to the surgery-alone group, which had a median recurrence-free survival of 10.7 months. Additionally, the median overall survival was shorter in the surgery group (33.9 months) than in the surgery-plus-HIPEC group (45.7 months). ²⁴ A cohort study involving 584 patients and a median follow-up of 42.2 months concluded that the group receiving CRS and HIPEC exhibited a longer median survival time (49.8 vs 34.0 months) and a higher 3-year overall survival rate (60.3% vs 49.5%) compared to the group undergoing debulking alone. ²⁵ Both studies noted that the addition of HIPEC to CRS did not result in higher rates of side effects, and the treatment was well-tolerated. Although several trials have been conducted to increase DFS or OS by adding HIPEC in ovarian cancer patients,^{24,26 -29} its widespread acceptance still needs more persuasive results. ³⁰

In this article, we have 2 primary objectives. Firstly, we will provide an overview of the molecular and cellular changes induced by hyperthermia that contribute to the enhanced efficacy of conventional therapies. Subsequently, we emphasize the impact of elevated temperatures on the immune system, as this secondary aspect significantly enhances the potential clinical application of hyperthermia.

The Molecular Effects of Hyperthermia

Many studies have been conducted to elucidate the molecular effects of hyperthermia, shedding light on factors influencing heat sensitivity or, conversely, heat resistance in both healthy and tumor cells. Physiological and molecular effects of hyperthermia are detailed in Figure 1. Mild hyperthermic therapy boosts blood flow, enhances tumor perfusion, and increases oxygenation, ³¹ thereby enhancing the efficacy of chemotherapy, radiotherapy, and the body’s immune response against tumors. Higher temperatures caused by heat shock hinder DNA repair mechanisms, ³² resulting in more replication errors, ³³ cell cycle arrest,^34,35 and apoptosis. ³³ However, the upregulation of heat shock proteins (HSPs) in response to protein damage and aggregation can induce thermotolerance, ³⁶ potentially counteracting the positive effects of hyperthermia. Thermal ablation triggers necrotic cell death, releasing tumor antigens and promoting antigen presentation, which activates immune cells. ³⁷ In this section, we will outline the molecular pathways activated during hyperthermia and have the potential to be used in the treatment of recurrent tumors.

Figure 1.

Physiological and molecular effects of hyperthermia. Mild hyperthermic therapy elevates blood flow, tumor perfusion, and oxygenation, thereby amplifying the effectiveness of chemotherapy drug delivery, radiotherapy, and the antitumor immune response. Elevated temperatures due to heat shock impede DNA repair mechanisms, resulting in an increased occurrence of replication errors, cell cycle arrest, and apoptosis. Conversely, the increased expression of heat shock proteins (HSPs) in response to protein damage and aggregation can induce thermotolerance, potentially counteracting the beneficial effects of hyperthermia. Thermal ablation initiates necrotic cell death, leading to the release of tumor antigens. This process facilitates antigen presentation and activates immune cells. Mild hyperthermia and heat shock also lead to immune response but in a non-necrotic way.

Inhibition of DNA Repair

Radiation therapy exerts its antitumor effect by inducing DNA damage. Often, it is combined with hyperthermia to enhance its efficacy by increasing the production of reactive oxygen species (ROS) and inhibiting DNA repair enzymes. Among the most severe forms of DNA damage is the double-strand break, which can be repaired through 3 mechanisms: non-homologous end joining (NHEJ), homologous recombination (HR), and the backup or alternative NHEJ process. ³⁸ NHEJ serves as the primary repair mechanism throughout the cell cycle, while HR is active exclusively during the S/G2 phase. HR utilizes the sister chromatid for error-free repair. However, when hyperthermia impairs the HR pathway, cells resort to NHEJ, a more error-prone method that leads to a higher occurrence of chromosomal rearrangements. ³² Hyperthermic treatment has demonstrated its effectiveness as a radiosensitizer by disrupting components of each repair pathway. ³⁹ Specific components of the HR pathway, such as the BRCA-1 and BRCA-2 proteins, ⁴⁰ are suppressed by heat shock, which increases the sensitivity of tumor cells to poly (ADP-ribose) polymerase 1 (PARP-1) inhibitors. ⁴¹ Additionally, heat disrupts the function of DNA-dependent protein kinase (DNA-PK), which is a crucial component of the NHEJ pathway. ⁴²

Cell Cycle Arrest

Mammalian cell division is meticulously controlled through checkpoints that monitor DNA integrity. One such pathway involves the activation of the ATM serine/threonine kinase (ataxia telangiectasia mutated), recruited to sites of DNA damage and targets various proteins, including well-known tumor suppressors like p53, BRCA1, and H2AX. ⁴³ The modification of histone H2AX was shown to be an important factor in heat-induced cell cycle arrest. The phosphorylation of this histone variant by ATM upon a double-stranded DNA break is known to be one of the first steps of the DNA damage response.^34,35 In MCF-7 human breast cancer cells, exposure to a 45.5°C heat shock triggers H2AX phosphorylation through dual mechanisms. Specifically, in the G1 and G2 phases, it induces double-stranded DNA breaks, while in the S phase, it causes replication fork arrest. ⁴⁴ Photoactivatable complexes initiate cell death in A549 cancer cells through a multifaceted process involving factors such as ROS-induced endoplasmic reticulum stress, mitochondrial dysfunction, DNA damage responses, immunogenic cell death, activation of PI3K/AKT signaling, and inhibition of cell growth in the S phase. ⁴⁵

The p53 protein plays a crucial role in stopping the cell cycle when it detects unrepaired DNA damage, especially before entering the S phase. ⁴⁶ Heat shock activates ATM, leading to phosphorylation of p53, ⁴⁷ leading to the increased expression of its downstream effector p21. ⁴⁸ p21 restrains cell cycle progression by inhibiting CDKs (cell-division cycle kinases) and DNA replication. ⁴⁹ An in vitro study on non-small cell lung carcinoma cells revealed that microwave hyperthermia, a potential post-operative treatment, hampers cell growth through G2/M cell cycle arrest via the ATM-Chk2-p21 pathway. ⁵⁰

The arrest in the G1/S phase appears to be contingent on the p53 status in specific cancer cell lines. ⁵¹ Cell lines with wild-type p53 exhibit robust activation of p21 and the G1/S checkpoint. Conversely, cells with mutated p53 display G2/M arrest upon heat shock. ⁵² Nevertheless, a p53-independent pathway for p21 activation has been documented in p53-deficient cell lines such as MDAH041 and T98G. ⁵³ Experimental data also highlight the impact of heat treatment duration and temperature on p53 phosphorylation status and the phase of the cell cycle at which arrests occur. ⁵⁴

The efficacy of both radiation and hyperthermic therapies varies across different phases of the cell cycle. S-phase cells exhibit increased radiation resistance due to elevated levels of DNA repair enzymes. However, their radiotolerance can be diminished due to heat-induced inhibition of these proteins. In the M phase, both DNA replication errors ⁵⁵ and heat shock ⁵⁶ induce abnormal mitosis. Heat disrupts the organization of microtubules, resulting in the formation of multipolar mitotic spindles and ultimately leading to cell death through centrosome destabilization. ⁵⁷ In MCF7 breast cancer cell lines, the 24-hour treatment interval has the most pronounced effect on the populations of cells in the S and G2/M phases, hence, this time frame is recommended for combined radiotherapy following hyperthermia treatment. ⁵⁸ Others found that this interval should be as short as possible for an optimal effect in experimental tumors, ⁵⁹ and in advanced cervical cancer. ⁶⁰ HSP70-linked thermal resistance is the highest at 24h, ⁶¹ and clinical hyperthermia protocols propose a minimum of 48 hours between the treatments. ⁶²

Induction of Apoptotic Cell Death

Apoptosis is a finely orchestrated mechanism responsible for regulating cell turnover and preventing the accumulation of harmful cellular abnormalities. Dysfunctional apoptotic processes are often observed in cancer cells, which gives them the hallmark of resistance to programed cell death. ⁶³ The hyperthermal induction of apoptotic cell death can be attributed to several potential mechanisms. First, rising temperatures induce protein unfolding and aggregation to such an extent that it exceeds the capacity of HSPs. This accumulation of unfolded proteins leads to endoplasmic reticulum stress, ⁶⁴ ultimately triggering cell death. ⁶⁵

Secondly, elevated temperatures can cause different types of DNA damage, including base damage, single-strand breaks, and inhibition of replication. ³³ The responses to DNA damage are primarily mediated by either the ATM-Chk2 or the ATR-Chk1 pathways. In human leukemia Jurkat cells, heat stress primarily activates the ATR/Chk1 pathway, leading to the activation of the G2/M checkpoint, cell cycle arrest, and subsequent apoptosis. ³³

Thirdly, hyperthermia has been shown to activate both the intrinsic and extrinsic signaling pathways of apoptosis induction. Heat shock influences 2 independent mechanisms within the intrinsic pathway: caspase-2-Bid and Bim-Bax/Bak. Hyperthermal treatment activates caspase-2, leading to the cleavage of Bid. ⁶⁶ The truncated Bid (tBid) initiates the mitochondrial pathway, involving alterations in the permeability of the mitochondrial outer membrane, the release of cytochrome C, and the formation of the Apaf-1-caspase-9 apoptosome. ⁶⁷ However, it’s noteworthy that the caspase-2-Bid pathway doesn’t solely account for heat-induced apoptosis. Instead, the activation of the Bim-Bax/Bak proteins has emerged as another mechanism for activating the intrinsic pathway upon heat shock. ⁶⁸ On the extrinsic pathway, apoptosis initiation occurs via cell surface death receptors. ⁶⁹ Elevated heat has been demonstrated to affect several receptor-mediated pathways, including Fas, ⁷⁰ TRAIL ⁷¹ and TNFα signaling. ⁷² Furthermore, the combined treatment involving hyperthermia and the use of the unripe fruit of the mandarin tree, Ponciri Fructus Immaturus (PF), a medicinal herb, synergistically suppresses the proliferation of AGS gastric cancer cells by inducing both extrinsic and intrinsic apoptotic pathways. ⁷³ However, the utilization of these natural herbs in Western clinical practice is uncommon due to challenges related to accurately dosing the active ingredients they contain.

Heat Shock Proteins (HSPs)

Heat shock proteins (HSPs) are cellular proteins synthesized in response to stressful conditions, ⁷⁴ such as elevated temperatures. When cells are exposed to high temperatures, they undergo a stress response to protect themselves from damage. ⁷⁵ These proteins help to stabilize and rebuild damaged proteins, prevent protein aggregation, and facilitate the removal of damaged proteins from the cell ⁷⁶

Heat shock proteins (HSPs) constitute a substantial family of chaperones, categorized by their molecular weight into HSP27, HSP40, HSP60, HSP70, HSP90, and the larger HSPs. ⁷⁷ These HSPs play pivotal roles in maintaining cellular homeostasis, encompassing functions such as protein folding, processing, and trafficking. They also facilitate the proteasomal degradation of faulty proteins ⁷⁸ and convey potent anti-apoptotic signals that enhance cell survival. ⁷⁹

The immunomodulatory impact of hyperthermia can be attributed to the increased expression of HSPs provoked by elevated temperature stress signals. While most HSPs are typically expressed intracellularly, there is mounting evidence of their presence outside of cells. ⁸⁰ Earlier studies suggested that extracellular HSPs may serve as danger signals for innate immune cells,^81,82 but recent findings indicate their potential anti-inflammatory effects. ⁸³ For instance, HSP70 has been shown to translocate to the plasma membrane in response to stress signals ⁸⁴ and the subsequent release of HSP70-containing membrane vesicles triggers the production of cytokines and NOS in macrophages. ⁸⁵ Conversely, more recent publications emphasize the immunosuppressive and regulatory functions of HSP70. ⁸⁶ The observed increase in inflammatory cytokine production may have been attributable to endotoxins or other contaminants. ⁸⁷ However, HSP70 promotes the differentiation of tolerogenic dendritic cells ⁸⁸ and regulatory T cells. ⁸⁹ A systematic assessment of experimental data has refuted claims that these cellular responses were merely due to contaminants from protein expression hosts, highlighting the context-dependent nature of HSP-mediated immunomodulation. ⁹⁰ Clinical hyperthermia could serve as a method to stimulate the immunological functions of HSP70 by increasing its expression and promoting its release through localized necrosis. ⁹¹ Sub-ablative heating, when combined with the clinically significant HSP90 inhibitor NVP-AUY922, could synergistically trigger a pro-immunogenic type of cell death in colon cancer cells. ⁹² The elicited immunological response can be influenced by the specific HSP family, whether they are expressed intracellularly or extracellularly, and the type and activation status of interacting immune cells. ⁹³

Alterations in HSP expression are commonly observed in various types of cancer, as these proteins play a crucial role in conferring resistance to environmental stressors, including reactive oxygen species (ROS), hypoxia, heat, and therapeutic agents. ⁹⁴ In mammals, heat shock factor 1 (HSF-1) serves as the principal transcription factor responsible for inducing the expression of HSPs, ⁹⁵ However, it has been revealed that it also regulates genes involved in host defense, inflammation, and cell survival. ⁹⁶ Elevated levels of HSPs provide a distinct advantage to tumors, and the overexpression of specific HSP families is often suggested as a biomarker for cancer prognosis. ⁹⁷ The presence of HSPs promotes cancer cell proliferation and invasiveness while concurrently aiding cell survival by inhibiting apoptosis. ⁹⁴ Consequently, numerous researchers strive to identify mechanisms to inhibit HSP expression without disrupting the homeostasis of healthy cells, where HSPs also fulfill vital functions. ⁹⁸

Despite the generally supportive role of most HSPs in tumor progression, specific members of the HSP40 family, such as HLJ1, ⁹⁹ Tid1 ¹⁰⁰ and DNAJB6 ¹⁰¹ demonstrate antitumor effects, further emphasizing the functional diversity within this family. Moreover, as discussed earlier, the immunomodulatory effects of HSPs can also be influenced by the activation status of immune cells. The concept of “hot and cold” tumors has emerged to describe the disparity in the tumor microenvironment (TME) between responder and non-responder mice. ⁶² These tumor types differ in terms of the number and composition of infiltrating cells, as well as the activation status of immune cells, which can significantly impact treatment efficacy. ¹⁰² Hyperthermal therapy has the potential to shift the phenotype of immune cells toward activation, priming them to respond to the presence of HSPs in an inflammatory and antitumor manner. This phenomenon helps overcome the suppressive nature of TME.

Induction of Thermotolerance

Clinical studies have revealed that the application of fractionated hyperthermia can lead to the development of thermotolerance when mild heat treatment is administered during subsequent sessions. ¹⁰³ Although this tolerance is temporary, it should be considered when determining treatment intervals. The precise molecular mechanisms underlying thermotolerance remain incompletely understood, but numerous articles suggest that it can be partially attributed to the heat-induced expression of HSPs. In HeLa cells, thermotolerance is induced by pre-treating them at 42°C before subjecting them to a 45°C heat shock. This process activates the JAK-Stat signaling pathway, resulting in increased expression of HSP27, HSP70, and HSP105. ³⁶ In nasopharyngeal carcinoma cells, inhibiting both HSP90 and HSP70 sensitized cells to heat-induced apoptosis, ¹⁰⁴ indicating that targeting HSPs could be a therapeutic strategy to combat thermotolerance. ¹⁰⁵

A common characteristic of heat shock is the upregulation of pro-apoptotic factors. Thermotolerance acquired at mild temperatures (40°C) leads to increased expression of anti-apoptotic members of the Bcl-2 family, ¹⁰⁶ inhibits caspase activation, ¹⁰⁷ and suppresses the apoptotic pathway mediated by the FasL death receptor. ¹⁰⁸ Another mechanism for cell survival in response to heat shock is autophagy. Autophagy safeguards cells by breaking down dysfunctional or aggregated proteins that are formed due to heat-induced damage and unfolding. ¹⁰⁹ The expression of autophagy-related proteins increases after heat shock, and this increase is even more pronounced in thermotolerant cells that were pre-treated at 40°C before undergoing a 42°C heat shock. ¹¹⁰ On the other hand, heat treatment-induced autophagy promoted invasion and metastasis through TGF-β2/Smad2-mediated epithelial-mesenchymal transition in a xenograft mouse model. This suggests that suppressing autophagy might be a suitable strategy to counteract tumor progression and metastasis following inadequate microwave ablation (MWA). ¹¹¹

Hyperthermia and the Immune System

Fever and the Immunomodulatory Effects of Mild Hyperthermia

Fever is a mechanism used by homeothermic organisms to combat infections or injuries by elevating their core body temperature.^112,113 It shares common features with the evolutionarily conserved heat shock response, which the body initiates in response to excessive environmental heat or stressors that can cause protein denaturation. Fever is induced when the activation of innate immune cells affects the nervous system. ¹¹⁴ Upon detecting pathogens, immune and endothelial cells release pyrogenic cytokines (such as IL-1, IL-6, and TNF-α), ¹¹⁵ After recognizing the pathogen, immune and endothelial cells produce pyrogenic cytokines (IL-1, IL-6, and TNF-α). ¹¹⁶ The presence of PGE2 and IL-6 maintains febrile temperatures by stimulating thermoregulatory neurons expressing the PGE2 receptor EP3 in the hypothalamus. ¹¹⁷ The stimulation of neurons by PGE2 results in increased heat generation in brown adipose tissue ¹¹⁸ and vasoconstriction in the extremities to minimize heat loss. ¹¹⁹ This rise in body temperature and metabolic rate aids organisms in overcoming infectious diseases.

Mild hyperthermia offers a similar benefit by inducing fever-like temperatures. While elevated body temperature can inhibit the proliferation of certain microbes,^113,120 its protective effect is primarily due to the enhanced activation of both innate and adaptive immune cells (Figure 2). Innate immune cells are thought to play a crucial role in initiating an antitumor response under fever-like hyperthermal conditions (39°C-41°C). The increased temperature stimulates a more robust respiratory burst ¹²¹ and encourages the infiltration of tumors by neutrophil granulocytes. ⁶ It also enhances the phagocytic and bactericidal capabilities of macrophages. ¹²² Dendritic cells (DCs), a vital antigen-presenting cell type in initiating an antigen-specific T-cell response^120,123 are also influenced by environmental temperatures. After antigen uptake, DCs migrate to lymph nodes to present the processed immunogenic peptides to naive T cells, during which they go through maturation. ¹²⁴ Hyperthermic treatment-induced DC maturation results in an increased expression of CD80, ¹²⁵ CCR7, ¹²⁶ and CD86, ¹²⁷ enabling their migration to lymph nodes for antigen presentation. ¹²⁸ However, it’s worth noting that some studies have reported an immunosuppressive effect of heat stress, particularly at temperatures above 41°C. ¹²⁹ Given that the immunological impact begins to occur at a temperature threshold of 40°C, ¹³⁰ it is crucial to meticulously determine the induced core temperature in therapeutic interventions. ¹²²

Figure 2.

The immunomodulatory effect of hyperthermia. Therapy-resistant tumors often employ strategies to evade the antitumor immune response, resulting in the creation of an immunosuppressive microenvironment. This environment is characterized by the prevalence of tolerogenic dendritic cells (DC), M2 macrophages, and regulatory T cells (Treg). However, mild hyperthermia can counteract these immune-suppressing mechanisms by enhancing the infiltration and activation of immune cells. Mild hyperthermia also promotes neutrophil degranulation, phagocytosis, and antigen presentation in M1 macrophages and dendritic cells. Following the uptake of antigens, dendritic cells migrate to lymph nodes, where they present tumor antigens to T cells. This process leads to the clonal expansion of cytotoxic T cells (Tc), which subsequently infiltrate tumors. Alongside natural killer cells (NK), these cytotoxic T cells work to eliminate cancer cells by releasing cytotoxic granules and activating the Fas-FasL pathway.

The initial activation of myeloid cells described above is followed by the development of an adaptive immune response. Elevated temperatures aid in lymphocyte priming by enhancing their adhesion and extravasation through high endothelial venules, dependent on L-selectin and α4β7 integrin. ¹³¹ This promotes homing to secondary lymphoid tissues, where the clonal expansion of antigen-specific T and B cells can occur. ¹³² Hyperthermia also enhances antigen presentation and T cell priming in an HSP70-dependent manner, ¹³³ resulting in an improved cytotoxic response. ¹³⁴ The infiltration of CD8+ cytotoxic T lymphocytes into tumors is a positive prognostic marker associated with improved survival. Therefore, many therapeutic approaches aim to boost this aspect of anticancer immunity. ¹³⁵ In a mouse model, local infrared radiation increased the number of activated dendritic cells in draining lymph nodes and the infiltration of T cells, B cells, and natural killer (NK) cells into tumors. ¹³⁶ Cytotoxic T cells eliminate their target cells either directly through secreted cytolytic factors or by inducing apoptosis via the Fas-FasL pathway. Hyperthermia has also been shown to enhance this facet of the T cell response, as evidenced by increased FasL expression and cytolytic activity of T cells. ¹³⁷

The downregulation of MHC I expression has been identified as an evasion strategy employed by various tumor types, including pancreatic, ¹³⁸ cervical, ¹³⁹ prostate, ¹⁴⁰ lung, ¹⁴¹ and others ¹⁴² to suppress antigen presentation and the cytotoxic T cell response. Reduced MHC I expression has been associated with lower numbers of tumor-infiltrating lymphocytes (TILs) ¹⁴³ and poorer patient survival rates. ¹⁴⁴ NK cells serve as a second line of defense against tumor cells by recognizing MHC loss. These cells express both inhibitory and activating receptors, and the integrated signal determines the immune response. In healthy tissues, MHC I molecules maintain NK cells in a resting state by binding to their inhibitory receptors. However, the loss of inhibition, along with the presence of activating tumor-associated ligands (such as MICA, MICB, and ULBP), triggers a cytotoxic response against tumors with low MHC expression. ¹⁴⁵ Clinical studies involving cancer patients who underwent hyperthermia treatment have demonstrated an increased number of NK cells and enhanced cytotoxicity. ¹⁴⁶ In one study involving 15 patients with prostate adenocarcinoma treated with transrectal microwave hyperthermia, NK cells isolated from their blood exhibited elevated cytotoxic activity, particularly during the 2-month follow-up visit. Interestingly, this enhanced immune activation was not observed in another group of 15 patients with benign prostatic hyperplasia who received the same treatment regimen. ¹⁴⁷ In patients with hepatocellular carcinoma, NK cells were the first to respond to local liver heating during a 60-minute hyperthermia treatment, displaying increased activity as early as 20 minutes after treatment initiation. The extent of NK cell activation in response to hyperthermia varied among patients, but those who demonstrated a significant increase later reported an improvement in their quality of life. ¹⁴⁸

These clinical findings were confirmed by in vivo studies using mouse models. ¹³⁶ Hyperthermic treatment was associated with enhanced NK cell activation and infiltration. ¹⁴⁹ Furthermore, fever-like temperatures were shown to indirectly influence the cytotoxic status of NK cells by upregulating the expression of activating ligands, such as MICA ¹⁵⁰ or ULBP, ¹⁵¹ in cancer cells. When damaged tumor cells release immunogenic peptides that are internalized and presented by dendritic cells, it has the potential to prime T cells for a response. Consequently, the infiltration of NK cells into tumors represents a critical aspect of an anticancer immune response and is associated with a positive prognostic value, as observed in colorectal ¹⁵² and gastric ¹⁵³ carcinoma.

The Combination of Immunotherapy With Hyperthermia

The immune system constantly surveils the body to prevent the proliferation of malignant cells. However, cancerous cells sometimes develop mechanisms to evade detection. ¹⁵⁴ A significant breakthrough in cancer therapy has been the concept of modulating the immune system to counteract the immunosuppressive effects of tumors. Over the past decade, monoclonal antibodies targeting checkpoint molecules in the T cell activation pathway, such as CTLA-4 and PD-1/PD-L1, have gained FDA approval for treating various types of tumors. Surprisingly, when these agents were used as monotherapy, only a small percentage of patients achieved a lasting response. ¹⁵⁵ Current clinical studies are focused on combining immune checkpoint inhibitors (ICIs) with other treatments, such as chemotherapy, radiotherapy, or VEGF/VEGFR-targeted therapy. ¹⁵⁶

While ICI therapies show promising results, there have been reports of severe adverse side effects ¹⁵⁷ and acquired resistance ¹⁵⁸ that limit treatment effectiveness. Combining ICIs with hyperthermia could offer a viable alternative with low toxicity. One of the advantages of hyperthermic treatment is its ability to modulate the tumor microenvironment (TME). Hypoxia within the TME contributes to the suppression of the antitumor immune response. ¹⁵⁹ Local heating of tumors can increase oxygen levels and enhance lymphocyte infiltration through improved blood flow.

The benefits of combining mild hyperthermia with anti-PD-1/PD-L1 therapy were demonstrated in a mouse model of pancreatic cancer. This tumor type is typically unresponsive to ICI treatment due to its extensive fibrotic matrix and immunosuppressive environment. Elevated temperatures reduced hypoxia and facilitated the recruitment of immune cells to the tumors, resulting in suppressed tumor growth and fewer metastatic nodes. ¹⁶⁰ Another mouse study showed that the combined use of anti-PD-L1 and photothermal therapy increased the number of tumor-infiltrating cytotoxic T cells and inhibited the growth of primary and distant tumors formed by the 4T1 breast cancer cell line. ¹⁶¹ In tumor-bearing mice, mild photothermal therapy led to substantial PD-L1 gene knockdown, reversed immunosuppression within the tumor microenvironment, promoted heightened anti-tumor immune responses, and effectively curtailed melanoma growth. ¹⁶² This also supports the idea that the direct immunomodulatory effects of hyperthermia, as discussed in the previous section, could potentially enhance ICI treatment. Additionally, NK cells were found to recruit dendritic cells to tumors, ¹⁶³ a crucial step for initiating a T cell response. ¹⁶⁴ Thus, the heightened activation and cytotoxicity of NK cells following hyperthermia also contribute to a more effective T cell response. In another study, administering dendritic cells (DCs) and modulated electro-hyperthermia (mEHT) directly into the tumor mass could enhance the antitumor effects of immunotherapy. ¹⁶⁵ It is essential to note that inducing a systemic immune response is crucial for the successful eradication of cancer. Immune cells activated at the tumor site can also target metastatic foci, thus maintaining a disease-free state.

Another approach involving hyperthermia is the precise destruction of solid tumors using high heat (>50°C) for a brief period, known as thermal ablation. Initially recommended for patients with solitary or oligometastatic tumors, its application in metastatic cancers is under active investigation due to its ability to induce a systemic antitumor response via the immune-activating nature of the debris remaining after cell death. ¹⁶⁶ Several ongoing clinical studies explore the use of thermal ablation as an adjuvant therapy to enhance the efficacy of immunotherapies. ¹⁶⁷ The advantage of ablation over surgical resection is that even though the tumor is destroyed, the remaining necrotic cell mass retains its antigenic properties for antigen-presenting cells. ⁸ Compared to apoptotic cells, necrotic cells were shown to induce a stronger immune response, including increased uptake of tumor cells, maturation, and expression of costimulatory molecules by DCs. ¹⁵³ As a result, there is a more efficient cross-presentation to T cells. ³⁷

In summary, hyperthermia has the potential to enhance the effectiveness of immunotherapy in cancer treatment. Numerous clinical trials have demonstrated the benefits of combining chemo- and radiotherapy with hyperthermia, while also confirming its high tolerability. ² Recent reviews have also examined the immune-modulating effects of radiotherapy alone or in combination with immunotherapy and hyperthermia. ¹⁶⁸

Nanoparticles and Immunotherapy

A wide range of nanoparticles, encompassing both organic and inorganic, are currently under scrutiny for in vitro and clinical applications Their diminutive size, ranging from 1 to 100 nm, coupled with a substantial surface area available for modifications, renders them well-suited for targeted delivery. ¹⁶⁹ Organic nanoparticles encompass a wide range of structures, including liposomes, polymers, micelles, dendrimers, and carbon nanotubes. ¹⁷⁰ These are frequently employed as drug delivery systems for chemotherapeutic agents. For instance, a PEGylated liposomal formulation of doxorubicin has been effectively administered in cases of solid tumors, breast, lung, and ovarian cancer. ¹⁷¹ Inorganic nanoparticles are manufactured from metallic or metal oxide materials such as gold, silver, platinum, or iron oxide. ¹⁷² Magnetic nanoparticles, in particular, have gained approval for various applications, spanning from imaging contrast agents, ¹⁷³ to targeted drug delivery, ¹⁷⁴ intratumoral hyperthermia, and photothermal ablation. ¹⁷⁵ A recent review provides a comprehensive overview of nanotechnology advancements and the biophysical aspects of nanomagnetism. ¹⁷⁶

Studies involving photothermal nanoparticles, which can be selectively directed to tumors and activated using magnetic or infrared radiation, exhibit promising outcomes in cancer treatment. ¹⁷⁷ These agents offer precise heat treatment administration without harming healthy tissues, holding potential for future clinical trials. ¹⁷⁸ Iron oxide nanoparticles, which have been utilized for treating iron deficiency anemia since the 1930s ¹⁷⁹ are still used today in modified forms such as iron oxide-carbohydrate complexes or colloids. ¹⁸⁰ Several studies have underscored the immunological effects of iron oxide nanoparticles, capable of triggering an antitumor response resulting in tumor regression. The FDA-approved iron-oxide particle, ferumoxytol, elicits a pro-inflammatory response from macrophages, leading to reduced tumor growth in vivo in mice. ¹⁸¹ In another mouse study, retention of nanoparticles was observed in tumors relied on the presence of innate immune cells, specifically neutrophils, monocytes, macrophages, and dendritic cells, which internalize these particles. This interaction was shown to be independent of particle surface modification and led to the infiltration of cytotoxic T-cells, delaying tumor growth. ¹⁸²

These magnetic nanoparticles also serve as an effective tool for targeted hyperthermia. As they accumulate in tumors, they deliver localized heat without causing harm to healthy tissues. For instance, the FDA approved NanoTherm iron-oxide particle for intratumoral thermotherapy in cases of recurrent glioblastoma in 2010 ¹⁸³ and for prostate cancer in 2018. ¹⁸⁰ In a study investigating the effectiveness of glioblastoma treatment, researchers combined the heating of injected nanoparticles with an alternating magnetic field and fractionated radiotherapy. This combined approach resulted in prolonged overall survival with manageable side effects. ¹⁸⁴

The combination of nanoparticles with immunotherapy is a burgeoning area of interest due to its potential to augment the immune response, both through the nanoparticles themselves and via the release of tumor-specific antigens following local ablation. PEGylated iron nanoparticles, when paired with immunotherapy, induced a systemic immune response and established immunological memory in tumor-bearing mice. This combination effectively controlled tumor metastasis and recurrence. ¹⁸⁵ In a similar model, the concurrent use of nanoparticle-mediated thermal ablation with ICIs facilitated the activation and maturation of dendritic cells, culminating in a robust cytotoxic T cell response and the eradication of distant metastases. ¹⁸⁶ Targeting the tumor microenvironments with nanoparticles has also been demonstrated as an effective strategy to overcome resistance to PD-1/PD-L1 blockade via ICI treatment in lung cancer patients. ¹⁸⁷

Magnetic hyperthermia in mice elevated the expression of the “eat me” signal, calreticulin, in 4TI breast cancer cells. This promoted their phagocytosis and triggered a cytotoxic T cell response, sensitizing tumors to anti-PD-L1 therapy. ¹⁸⁸ While the study did not report an increase in overall survival, it underscored the beneficial impact of hyperthermia when combined with immunotherapy in terms of local tumor control and the recruitment of T lymphocytes. ¹⁸⁹

The composition and surface properties of these nanoparticles are highly adaptable, enabling precise control of the elicited immune response, whether enhancing or avoiding it. ¹⁹⁰ Moreover, surface modifications with specific antibodies or ligands facilitate their precise delivery to tumors, ¹⁹¹ paving the way for the development of drugs with minimal off-target effects. In conclusion, the low toxicity and straightforward manufacturing process make nanoparticles a promising avenue for future research in cancer treatment.

HiFU and Immunotherapy

High-intensity focused ultrasound (HiFu) is a non-invasive, cutting-edge medical technology that utilizes focused beams of ultrasound energy to target specific tissues within the body, to induce hyperthermia in order to achieve therapeutic effects. ¹⁹² In the context of cancer treatment, HiFu is being explored as a promising alternative or adjunct to traditional therapies such as surgery, chemotherapy, or radiation.^193,194

The mechanism of action behind HiFu involves the conversion of ultrasound energy into heat as it passes through tissues. This localized hyperthermia can trigger various biological responses, including apoptosis, ¹⁹⁵ denaturation of proteins, ¹⁹⁶ and activation of the immune system ¹⁹² to recognize and eliminate cancer cells. T lymphocytes harvested from mice subjected to HiFu treatment demonstrate the ability to orchestrate cellular immune responses against tumors, suggesting a pivotal role of HiFu-induced immunomodulation in fostering anti-cancer immunity. ¹⁹⁷ HiFu therapy in veterinary patients can facilitate tumor growth inhibition, immune system activation, and enhance the efficacy of chemotherapy. ¹⁹⁸ HiFu initiated immune sensitization in resistant murine neuroblastoma, enhancing its responsiveness to checkpoint inhibitor therapy. ¹⁹⁹ HiFu effectively improved tumor microenvironment, eventually hindering tumor growth and metastasis by enhancing the host’s anti-tumor immune response following HiFu ablation. This synergy is particularly notable when combined with PD-L1 inhibitor-based immunotherapy. ²⁰⁰ In summary, HiFu appears to be a promising adjuvant tool in the battle against malignant diseases.

Outlook

Clinical studies have demonstrated that hyperthermia is generally well-tolerated. However, the efficacy of this treatment can vary depending on the type of cancer, and the choice of heating technique can have a significant impact on treatment outcomes. Due to the different depth penetrance of the discussed applications these lead to different outcomes at the same temperature. It has been observed that both the temperature and duration of heat treatment play a pivotal role in influencing various cellular responses associated with hyperthermia. This variability may stem from the wide range of effects attributed to hyperthermia in the literature, which can sometimes be contradictory. The decision between employing mild hyperthermia or heat shock, as well as the treatment duration, can affect the induction of autophagy, apoptosis, or necrosis, and determine the activation of specific cell cycle checkpoints. The response of immune cells can range from activation to inhibition. Therefore, optimizing temperature and heating duration becomes crucial for achieving beneficial immune modulation.

Cells have the ability to adapt to environmental stressors, such as cytotoxic drugs or extreme heat. Fractional hyperthermia treatment and other complementary applications such as radiotherapy or chemotherapy (especially when intervals are inadequately chosen) can lead to the development of thermotolerance, thereby diminishing its effectiveness. Among the key factors contributing to thermotolerance is the heat-induced expression of heat shock proteins (HSPs). Thus, targeting HSPs may offer a viable therapeutic approach.

Moreover, it is imperative to highlight the ongoing need for advancements in heating technologies and monitoring. Inconsistent heating can lead to a varied response, which may potentially induce thermotolerance in certain regions of the tumor and enable the growth of residual cells. The precise targeting and monitoring of heat administered to these tumor sites requires further development. The use of iron-oxide nanoparticles as imaging contrast agents has already been established and validated through in vivo studies for safety and effectiveness. Photothermal nanoparticles may offer an optimal solution for precisely targeting tumors that are deeply seated.