High hydrostatic pressure (HHP) is a traditional technology used to produce steel, alloys, ceramics, and synthetic materials (1). Over the past few decades, HHP has also been used for non-heat pasteurization of processed foods, designed to extend the storage time of foods, such as juice, milk, and canned products (2). As researchers in different scientific fields continue to explore HHP, some new applications of the technology are emerging (3,4). Since most practical applications of HHP technology are subjecting biological systems to hydrostatic pressure (HP), the uniqueness of this method is currently being investigated at various levels, ranging from viruses, enzymes, microorganisms, mammalian cells, and tissues (5–7). Usually, all pressures are causing a reduction in the volume of the system, which can lead to changes in its structure and disturb the equilibrium of chemical reactions (8). Excessive pressure may lead to the destruction of cell structure (9). For numerous thermophilic microorganisms, HP inhibits cell growth in the range of tens of megapascal (MPa) and completely inhibits cell growth at approximately 50 MPa (10). Pressure greater than 200 MPa can annihilate most microorganisms (11).

Cancer poses a huge threat to human health. Currently, the main cancer treatments are surgery, radiation therapy (RT), and chemotherapy (CT). Multimodal treatment strategies may be effective in reducing tumor size, regressing solid tumors, and extending patient life (12). However, the recurrence and metastasis of tumors gives rise to a poor prognosis for numerous patients (13). Therefore, the goal of oncology treatment is not only to exterminate tumor cells of the primary origin but also to obtain long-lasting antitumor effects in order to control metastatic and recurrent tumor cells. However, these treatments currently leave much to be desired in terms of providing sustained antitumor effects and limiting tumor metastasis and recurrence. In addition to these disadvantages, these treatments have numerous toxic side effects that affect normal tissues (14). Combining the antitumor effects of the body with the host immune system to generate an effective antitumor immune response is an attractive therapeutic approach.

Tumor vaccines are designed to specifically activate the immune system of patients (15). Therefore, it is necessary to activate the immune response of patients to the tumor. The immune system must be trained to control dormancy and metastasis of residual tumor cells (16). Tumor vaccines may bypass the complex processes of defining individual antigens. There are numerous associated antigens on the surface and inside the tumor cells that prevent tumors from escaping immunity (17). To enhance the antitumor immune response, additional application of immune adjuvants is beneficial (18). The combination of conventional therapies with immunotherapy may improve the overall patient survival. In addition, immunotherapy may be more appropriate for oncology patients because of its lower toxicity compared to CT.

HHP technology is an effective approach to the production of tumor vaccines. Helmstein reported the application of HHP for the treatment of bladder cancer in 1972 (18). The patients were treated with hydrostatic bladder dilation. Subsequently, some authors reported that vaccination of HHP-treated tumor cells treated with the chemical cross-linker adenosine dialdehyde alone or in combination with the reducing agent N-acetyl-L-cysteine induced antitumor immunity in vivo (19) and in vitro (20). In this review, we summarized the latest knowledge on the relevance of hydrostatic hypertension for immunotherapy of biomolecules and tumors and discussed possible future directions for the development of HHP tumor vaccines.

The physical property of pressure is defined as the force per unit area acting on the surface in a direction perpendicular to the surface: P=F/A in which P represents the pressure, F represents the normal force applied to the surface, and A represents the area of the surface. The official unit of pressure is the Pascal (Pa) (1 Pa=1 N/1 m²=10⁻⁵ bar). The Newton represents a small force, while 1 m² corresponds to a large surface, thus the Pascal is a very small unit of pressure. Therefore, MPa (1 MPa=10⁶ Pa=10 atm) is a common unit of pressure used in HP research. The conversion from MPa to other units of pressure is presented in Table I (21).

The first research on HP traces back to the late 19th century and was carried out mainly by oceanographers and physiologists (22). HP exists in all the explored biological environments. The pressure extends from 0.1 MPa (atmospheric pressure) at the sea level to 110 MPa at the deepest part of the ocean in the Mariana Trench, 11 km below sea level (23). The average depth of the ocean is 3.8 km. The average pressure on various marine organisms is approximately 38 MPa, which is 380 times the atmospheric pressure (24).

Roger first reported the use of HP technology to kill microorganisms such as Staphylococcus aureus and Escherichia coli in 1895 (25). Hite (26) and Hite et al (27) studied microbial inactivation in milk using 650 MPa HHP technology in 1899 and developed microbial inactivation for extended storage of vegetables and fruits in 1914. From 1932 to 1952, some researchers studied HHP technology to inactivate the different microorganisms for food processing and biological applications. For example, they studied the effects of HHP on bacteria (28), viruses (29,30), antigens (31), antibodies (31), and tumors (32). The effects of HHP technology on macromolecular and eukaryotic physiological processes have been extensively studied since the middle of the 20th century, mainly using sea urchins and frog eggs (33–35), as well as epithelial cells, chondrocytes, and tumor cells (36–38). In the early 1990s, HHP technology was further developed in Japan and used for the processing and preservation of food products without the thermal treatment and the addition of preservatives (39), since it does not affect vitamins and pharmacologically active molecules and does not change flavors and aromas (40). In addition to food sterilization and preservation, HHP technology has been widely used in numerous other applications (41). In the biotechnological and pharmaceutical industries, HHP technology is also used in the sterilization of bone grafts and in the development of tumor vaccines, all of which take advantage of the molecular effects of HHP (41).

Over the past few decades, a growing number of disciplines have begun to explore the potential of exposing a variety of complex biological units to HHP, including proteins, lipids, nucleic acids, eukaryotic cells, and multicellular tissues (41). In general, all pressure effects correspond to a reduction in the volume of the biological unit and the acquisition of a more compact structure (8). The aforementioned will be discussed in more detail in the later sections.

Cancer immunotherapy (CI), particularly when used in combination with other therapies such as RT and CT, is a promising avenue to cancer treatment. Shi et al have demonstrated the synergistic effects of the combination of CI and CT in the clinical study (103). CI stands out in the second-line treatment for recurrent tumors and metastases by activating the immune systems of patients to trigger an antitumor response (103). Since tumor recurrence and metastasis remain the main reasons for tumor-related deaths, the identification of tumor cell specificity and persistence will be the focus of future research. To date, the number and type of specific T cells required for the efficient antitumor therapy are unknown (104). Although different subtypes of immune cells are suspected to have various effects on tumor progression, the infiltration of immune cells is usually associated with the prognosis of most solid tumors (105,106). Therefore, CI is a suitable adjuvant to standard tumor therapies because it is designed to activate the immune system of the patient against tumor cells (107).

When developing multimodal concepts for tumor therapy, the treatment methods of tumor vaccines should be considered (108). Vaccination is an agent that causes the host to receive treated autologous tumor cells in order for a large number of defined tumor antigens to simultaneously stimulate the immune system of the host (109). HHP inactivation of tumor cells can be performed in a highly repeatable manner according to Good Manufacturing Practices (GMP) and legal requirements. These vaccines must also fulfill the major requirements for all cell-based therapeutic tumor vaccines, including i) complete inactivation of tumor cells, ii) maintenance of immunogenicity, and iii) compliance with statutory provisions (110). Physical (X-ray and freeze-thawing) and chemical methods have been used to inactivate tumor cells in vaccination experiments (46). However, these methods usually have some restrictions. Tumor cells cannot be inactivated safely by a mild treatment, or the inactivated cells are weakly immunogenic (46). HHP fulfills these specifications for clinical vaccine: It inactivates tumor cells effectively, is non-toxic, does not wreck the immunogenicity of the tumor cells, and can comply with GMP and legal requirements. In additiom, it is a repeatable and easy to apply method (111). Therefore, HHP treatment is superior to other methods, such as freeze-thaw, radiation, or heat killing methods. Furthermore, the HHP approach cannot mix other chemicals into tumor vaccines, unlike the chemical methods (49). These advantages suggest that HHP is a promising method for generating tumor vaccines (112).

The use of HHP in tumor inactivation and tumor vaccine development has been studied since the 1970s (113). In 1972, HHP was reported as the tumor treatment method and was investigated for the treatment of bladder cancer by using the hydrostatic bladder dilatation method (18). Later, Deckmann et al reported that treatment of leukemic cells with 150 MPa HHP resulted in enhanced immunogenicity (114). Since then, HHP has been used to inactivate tumor cells to develop vaccines. Eisenthal et al treated tumor cells with HHP (120 MPa) in the presence of the biocompatible crosslinker, adenosine dialdehyde (115). Treated cells were potent immunogens because HHP increases the antigenic presentation by rearranging the cell surface proteins into immunogenic clusters (118). This study indicated that HHP-killed tumor cells can trigger antitumor immune responses. This syngeneic tumor vaccine that mimics the autologous vaccine from their tumor cells should contain all relevant tumor-associated antigens (TAAs) that may target a specific patient (116).

The purpose of tumor vaccines is to train the immune system to actively develop lasting immune memory to fight the metastasis and recurrence of tumors (117). Weiss et al concluded that the inactivation of intact tumor cells induced by HHP, the degradation of the nucleus, and the preservation of the immunogenic potential of these dead tumor cells facilitate the use of this technology for the production of tumor vaccines (46). Frey et al have also revealed that HHP-treated tumor cells can preserve their shapes for more than a few weeks, which appears to be important for the production of potent vaccines (24). HHP treatment also caused a marked increase in cytoplasmic viscosity of the treated cells, which would lead to a slow and sustained release of cell-derived antigens and danger signals (45). These features are critical for the phagocytosis of tumor cells and subsequent presentation of cognate antigens by dendritic cells (DCs), and further expand the prospects of applying HHP technology to produce tumor vaccines (118).

Notably, HHP-induced cell necrosis occurs simultaneously with cytoplasmic gelation, and these cellular particles appear to maintain the relevant immunogenicity (42). Frey et al have proposed the use of HHP-treated tumor cells as the whole-cell-based tumor vaccines, due to the preservation of the antigenicity (24). Moserova et al revealed that HHP treatment could induce immunogenic cell death in tumor cell lines and revealed some molecular mechanisms associated with this phenomenon (119). Apoptosis induced by HHP treatment was controlled by the overproduction of ROS, which caused a rapid establishment of an integrated stress response and subsequent activation of caspase-2, caspase-3, and caspase-8 activation in dying tumor cells (119). Traditionally, apoptosis is considered immunologically silent, but specific immunogenic molecules, such as calreticulin, adenosine-triphosphate (ATP), HSP 70/90, or HMGB1, are released at or near the cell surfaces, thereby activating immune cells to enhance their antitumor activity (119,120).

Processing of tumor cells by HHP promotes the production of ROS. The processing of cells by pressure triggers downstream signaling pathways, such as cleavage of caspase-2, caspase-3, and caspase-8 (121). Moserova et al reported the application of various ROS scavengers and indicated that for HHP-induced calreticulin, the production of ROS was one of the prerequisites (119). More specifically, HHP-mediated ROS production may affect endoplasmic reticulum (ER) homeostasis, further triggered the phosphorylation of extension initiation factor (eIF)-2α and the cleavage of caspase-2, which was important for HHP-induced danger signaling involving calreticulin induction (122). Sandow et al have revealed that ER stress and ROS production may or may not lead to caspase-2 cleavage, depending on the prevailing environment (123). In addition, specific antibody blockade of calreticulin or depletion of caspase-2 significantly inhibited DC phagocytosis (119). While it remained unclear how caspase-2 regulated the exposure of calreticulin, the localization of this caspase in the ER and Golgi systems suggested the possible involvement in regulating the transit mechanisms (124). However, inhibition of ROS production was not sufficient to eliminate HHP-induced calreticulin exposure, suggesting that ROS-independent mechanisms may also be involved in this process. Therefore, exogenous calreticulin is important for the phagocytosis of tumor cells and the induction of specific immune responses in vaccine patients (125).

In previous studies, HHP technology was identified as an inducer of antitumor immunity in a variety of tumor cell lines (126–128). Due to its immunogenicity, this physical modality has been standardized and validated for incorporation into the manufacturing process of tumor immunotherapy products (129). In numerous cases, it was not possible to obtain autologous malignant cells from patients due to the inoperability of the tumor. The number of malignant cells in the biopsy or resected tumors was too small to produce a vaccine for the repeated administration during the immunotherapy. The HHP-killed tumor cell lines also retained an abundance of specific or tumor-associated antigens, which represented an advantage of this approach over numerous vaccines, where the number of antigens is often limited (130). Autologous tumor cells are a suitable source of antigens for vaccination. Providing an antigen array on the cell surface reduces the risk of tumor immune escape and eliminates the need to define individual antigens (131).

The advantages of vaccines based on autologous whole tumor cells are that they do not have to prospectively identify target antigens and can provide numerous TAAs that aberrantly express autoantigens. In contrast to neoantigens, the latter should only activate remaining low-affinity T cells and have to overcome the self-tolerance (132). Several additional methods have been developed to address the barrier, such as the addition of adjuvants, repeated vaccination, or co-stimulation (104). HHP-treated whole-cell-tumor vaccines have the advantage of providing multiple antigens and therefore lead to better results. This approach has been demonstrated in clinical trials in multiple myeloma (133) or renal cell carcinoma (134). Therefore, the inactivated form of whole tumor cells used as a vaccine, as well as the cell death induced in the primary tumor by standard methods, is important for triggering the effective antitumor immunity.

Large numbers of treated apoptotic tumor cells have been shown to trigger an effective antitumor immune response in mice (135). Galluzzi et al reported two morphologically equivalent but immunologically distinct subcategories of apoptosis, immunogenic and non-immunogenic apoptosis, thus introducing the concept of immunogenic cell death (ICD) (112). ICD was primarily mediated by defined spatiotemporal release or exposure of relevant danger signals or the damage-associated molecular patterns (DAMPs), which could act as adjuvants or associated danger signals to the innate immune system to trigger host protective antitumor immunity (136). Recently, several DAMPs have been associated with ICDs, where surface exposure of the ER-resident chaperone calreticulin was one of the main checkpoints for determining cellular immunogenicity (137). McDaniel et al reported that tumor cell ICDs were characterized by induction of ER stress response, production of ROS, and release of danger-related molecules, such as calreticulin, HSP, HMGB1, or ATP (138). Several tumor chemotherapies and cell physical death-inducing modalities have been described to induce ICD of tumor cells (139).

HHP is a convenient way to inactivate tumor cells and maintain immunogenicity (49). Fucikova et al reported that HHP treatment with 250 MPa induced ICD in human acute lymphocytic leukemia, prostate, and ovarian cancer cell lines, and primary tumor cells (140). HHP-induced ICD in tumor cells exhibited molecular characteristics similar to those induced by anthracyclines (141), such as induction of endoplasmic stress response and ROS formation, cell surface exposure of HSP and calreticulin, and release of ATP and HMGB1 (122). Physical cell death induction modalities, such as HHP or heat treatment (HT), have been demonstrated to be used as vaccines and to help induce antitumor immunity in patients. These modalities, especially HHP treatment, are effective inducers of ICD in malignant cells and may have a great potential in the development of new DC-based or whole cell-based vaccines.

Urbanova et al reported that lung cancer cells treated with 150, 200 and 250 MPa HHP exhibited a distinct ICD-induced temporal pattern, but incubating them with DCs for 24 h equally stimulated the expression of maturation-related and co-stimulatory molecules (130). When tumor cells were treated with ICDs, they activated various immune cells to stimulate antitumor immune responses (142). These findings demonstrated the significant role of the immune system in the antitumor treatment. Nevertheless, more research is required on the molecular mechanism of HHP and HT-induced ICDs, as well as on the CT drugs and radiation currently used, to optimize the treatment strategies. Future research should strive to incorporate the design of novel modern ICD-inducing agent-based immunotherapies into current multimodal oncology treatment regimens.

DCs play a key role in the immune response because they can capture antigens bound to the pattern recognition receptors, process and present them to the naive T cells, thus inducing the T-cell activation and thus creating an important bridge between the innate and adaptive immune systems (143). DCs have been demonstrated to play a crucial role in the induction of antitumor immune responses (119). DC-based immunotherapy is safe and induces antitumor immunity in patients with advanced melanoma (144). De Sanctis et al revealed that in an orthotopic mouse model of prostate cancer, the experimental group demonstrated that DC-based vaccines were as effective as CT in slowing tumor growth (145). Mikyskova et al demonstrated that DC-based vaccines were a reasonable tool for treating human prostate cancer (126). Hradilova et al reported important preclinical data from phase I/II clinical trials in non-small cell lung cancer (NSCLC) using DC-based active cellular immunotherapy in combination with CT and immune boosters for the treatment of NSCLC (127). Urbanova et al also reported important preclinical data from an ongoing phase I/II clinical trial in NSCLC involving the use of DC-based active cellular immunotherapy to produce immunogenically-killed lung cancer cells (130). These studies demonstrated that a DC vaccine can lead to long-lasting tumor immunity, a process that requires three steps (146). In the first step, DCs must obtain the relevant TAAs. Secondly, DCs have to mature and induce T-cell responses. In DC-based cellular immunotherapy, the enhanced ability of DCs to co-stimulate naive T cells during maturation may be important. The final step is to allow T cells to overcome the immunosuppression of the primary solid tumor and enter the tumor bed (146).

The success of DC-based tumor immunotherapy depends on the range of TAAs presented by DCs and the ability of DCs to produce cytokines such as IL-12p70 and provide the co-stimulation to T cells (147). Immature DCs are constantly migrating in the tissues and blood, scanning the environment for danger signals or potential pathogens (148). These pathogens and signals can activate innate immunity and interact with pattern recognition receptors, purinergic receptors, and phagocytosis-related receptors expressed by DCs and stimulate the presentation of tumor antigens to T cells (148). Typically, autologous DC-based vaccines use in vitro cultures of DCs loaded with the tumor antigens and promote the maturation of the DCs (149). Tumor cells are phagocytosed after being recognized by DCs, which then undergo antigenic processing. DCs must reach the mature stage to induce an effective immune response because semi-mature DCs have tolerogenic features (146). The maturation of DCs is accompanied by a decrease in antigen assimilation and an increase in migration capacity. DCs move to the lymph nodes (LN), where the complex of peptide and MHC-class II is presented to the antigen-specific T-cell receptor (TCR) on the naive CD4⁺ T-cell. This again indicates the enhanced DCs function in tumor patients (150).

Tumor cells can be inactivated by different methods and the choice of the optimal inactivation method is crucial for the DC vaccine optimization (15). HHP treatment has been revealed to maintain immunogenic tumor cell inactivation. HHP-treated tumor cells can induce the monocyte-derived DC maturation. DCs cultured with HHP-treated tumor cells can also induce the activation of T cells in vitro (49). Phagocytosis of HHP-killed tumor cells by DC stimulates the expression of maturation-associated molecules on DCs and induces the production of proinflammatory cytokines. The tendency of increased numbers of the CD8⁺ T cells and the natural killer (NK) cells in the spleens of the experimental animals are detected when DCs are pulse-stimulated with HHP-treated tumor cells (126,130).

Fucikova et al reported that the interaction of immature DCs with HHP-treated tumor cells resulted in the increased uptake of tumor cells by DCs and induced the expression of maturation-related molecules on DCs and the production of IL-12p70 and the related proinflammatory cytokines, suggesting that HHP-treated tumor cells provided an effective activation stimulus to DCs (140). Human monocyte-derived DCs pulsed with HHP-treated tumor cells, showing increased expression of maturation-related molecules and the production of the pro-inflammatory cytokine, resulting in the stimulation of CD4⁺ and CD8⁺ T cells produced by interferon-γ (IFN-γ) in vitro (140). These results suggested that despite few antigens detected by the western blot test, a significant amount of antigens were still present in DC-processed cells and presented in the major histocompatibility complex (MHC) class I molecules to CD8⁺ T cells (127). Hradilova et al revealed that a DC-based HHP-treated lung cancer vaccine produced by monocytes from NSCLC patients induced antigen-specific CD8⁺ and CD4⁺ T cells (127). A DC-based vaccine combined with the HHP-treated transgenic adenocarcinoma of the mouse prostate tumor cells combined with docetaxel CT significantly reduced tumor growth in each mouse model (151). These encouraging results revealed that HHP can be a significant method for tumor cell inactivation.

HHP treatment for tumor cells can be standardized according to GMP requirements and incorporated into manufacturing protocols for DC-based cellular CI (130). More importantly, DCs loaded with HHP-treated tumor cells exhibited enhanced phagocytosis, expressed high levels of co-stimulatory molecules, and stimulated a large number of specific T lymphocytes, whereas no T regulatory cells were induced in the absence of the additional immunostimulatory agents (140). Mature DCs express high levels of co-stimulatory molecules and peptide-bound MHC class I and II molecules and produce pro-inflammatory cytokines, which are essential for efficient stimulation of tumor antigen-specific T-cell responses (152). These interactions effectively activate DCs to phagocytose dying tumor cells and allow them to mature and acquire an immunostimulatory phenotype (152). A schematic diagram of DC-based vaccine preparation using immunogenic HHP-treated tumor cells, which can be applied to other physical tumor cell death-inducing modalities is presented in Fig. 3.

Phospholipids, such as lysophosphatidylcholine (LPC) and phosphatidylserine (PS), are involved in the clearance of apoptotic and necrotic cells. Apoptotic exposure of PS is one of the main ‘eat me’ signals for macrophages (153). Annexin A5 (AnxA5), a high specific ligand for PS, is an important modulator of immune responses against the PS-exposed particles (154). AnxA5 is also the focus of therapeutic applications for the delivery of drugs to the relevant cells expressing PS on the cell surface (154). AnxA5 binds to phospholipids in a Ca²⁺ dependent manner and it blocks the phagocytosis of dying tumor cells by macrophages, but not DCs. Thus, the clearance of dying tumor cells is transferred from macrophages to DCs (155).

PS also expose the luminal surfaces of the vascular endothelium of tumors. In animal models, antibody-targeting of PS damages tumor vasculature and induces antitumor immune responses (156). In addition in vivo, the provided AnxA5 decreased the uptake of apoptotic cells by the peritoneal macrophages and increased their uptake by DCs (157). In summary, AnxA5 promotes DC uptake by interfering with the macrophage-mediated clearance of apoptotic and necrotic tumor cells. In the presence of AnxA5, the microenvironment becomes inflammatory and leads to the regression of allogeneic tumors as well as the rejection and cure of the syngeneic tumors. The influence of AnxA5 on clearance of apoptotic tumor cells and antitumor immunity is presented in Table III.

The pattern of tumor cell death, whether induced by treatments in vivo or by inactivation of tumor cells in vitro for vaccine preparation purposes, has made an important contribution to the efficacy of antitumor immune responses (Fig. 4). The synergy of immunotherapy and RT has the potential to provide better local tumor cell targeting by providing better tumor control in non-irradiated areas (158). Individual differences in response to standard tumor therapies are usually observed clinically, ranging from complete remission to treatment progression. RT, CT, and AnxA5 are all considered as the immune modulators of tumors (159). They change the tumor cell phenotype early after the application. Exposure to stress proteins such as HSP70 and phagocytic recognition molecules such as PS can kill tumor cells through apoptosis or necrosis (160). The latter exists in a programmed and accidental form. Necrotic cells lose membrane integrities, leading to the release of immune-activated DAMPs, such as HMGB1, ATP, or HSP70, while apoptotic cells maintain membrane integrity and DAMPs are hidden (161). Apoptotic cells are cleared and recognized rapidly by PS, and macrophages release anti-inflammatory cytokines, resulting in an immunosuppressive microenvironment (162). Conversely, DAMPs mature and activate DCs, thereby promoting the cross-presentation of tumor cell-derived antigens with T cells.

In addition, DAMPs may also directly activate cells of the innate immune systems, such as NK cells (163). Inhibition of apoptotic cell clearance by macrophages with AnxA5, or induction of abundant apoptotic cells in a multimodal treatment setting, can promote the necrotic immune form of tumor cells (164). Immunogenic forms of tumor cell death can also be induced by killing biopsy-derived fresh tumor cells in vitro, resulting in complete cell death by increasing the immunogenicity (165). Fig. 5 summarizes the principle of multimodal treatments to induce tumor cell death leading to antitumor immunity.

The purpose of this short review was to summarize the knowledge and applications of HHP technology in the development of tumor vaccines and to envisage new possible research directions and applications. HHP technology has great potential for the development of tumor vaccines and provides a new treatment scheme for cancer patients (Table IV). Achieving the sustained antitumor response is a major limitation of most current therapeutic methods of solid tumors and additional and multimodal treatment approaches are required. HHP technology is an effective method for producing whole-cell vaccines or DC-based antitumor vaccines. However, to date, studies of both vaccines remain in the preclinical phase (41). Therefore, there is still a need to develop new vaccines for treatment with HHP that can avoid the disadvantages of existing HHP vaccines and activate the immune system to produce sustained antitumor immunity (Table V).

Tumor vaccines require activation of T cells to resist the immunosuppressive microenvironment (166). Progressive tumors usually promote tumor growth by promoting infiltration of tumor-bearing M2-like macrophages, myeloid-derived suppressor cells (MDSC), and regulatory T (T-Reg) cells, thereby inhibiting the local expansion and effector functions of CD4⁺ T helper cells and cytotoxic CD8⁺ T cells (167). Reluctant T-cell transfer vaccines with or without costimulatory antibodies, particularly against CD27, CD40, and CD137, can expand the tumor-specific T-cell pool (168). In most patients, T cell-mediated immunity in the tumor microenvironment is affected by several mechanisms of suppressive immune cell use within the tumor as well as by T-cell checkpoint suppression (169). This converts T cells into lymphocytes, which may have a transient but modest effect on the tumor. Specialized and selective regulation of the microenvironment may lead to temporary tumor shrinkage and render intra-tumor T cells resistant to the tumor (170). This may be achieved by inducing an acute inflammatory response using pattern recognition receptor (PRR) agonists or by removing or inhibiting regulatory mechanisms that modulate immunity [T-Reg cells, MDSC, and/or M2 tumor-associated macrophages (TAM)] (171). If there is no additional activation of a strong tumor-specific T-cell response, there is no significant effect on tumor growth in most patients (172). If a tumor-specific T-cell response has been ignited, checkpoints against cytotoxic T-lymphocyte-associated protein 4, programmed cell death protein 1, lymphocyte activation gene 3, antibody to T-cell immunoglobulin mucin receptor 3, or natural killer cell receptor A (or against their respective ligands) can help maintain the full effector function of T cells within the tumor (173). In some patients, this will lead to tumor destruction, while in others, immunosuppression of cells in the microenvironment may prevail (174). In situations where immunosuppression is alleviated and the tumor is sufficiently immunogenic, activation of DCs and M1-like TAMs will promote the attraction and activation of tumor-specific T cells and maintain the antitumor activity over time (175). They may lead to tumor eradication.

Several studies have observed in groups of patients with different types of tumors that patients have improved outcomes with immunotherapy when B cells compose a cluster of cells called tertiary lymphoid structures (TLS) within the tumor (176,177). Tumor-infiltrating B lymphocytes have been found in some tumor tissues and are an important component of TLS in tumor tissues (178). Tertiary lymphoid structures are ectopic lymphoid organs formed in non-lymphoid tissues during chronic inflammatory as well as tumor formation and consist of T cells, B cells, follicular dendritic cells, as well as other cells (179). TLS can be present in tumor tissue in various states of maturation, with the highest level forming germinal center structures (180). The impact of tumor-infiltrating B cells and TLS on tumor formation and the efficacy of immunotherapy have also received attention (180), but their specific roles in tumors and their underlying mechanisms are not fully understood. These results also indicate new directions for subsequent research, combining T cell-mediated immunotherapy with approaches using B cells, which may lead to more effective antitumor therapies for more patients.

Inactivation technologies used to prepare tumor cell vaccines should be aimed at inducing immunogenic malignant cell death forms. If tumor cell vaccines are prepared by the inactivation of whole tumor cells, the immunogenicity of the dead tumor cells should be enhanced or at least maintained by this procedure. The main focus of future oncology treatment concepts should be to combine classical antitumor therapy with immunotherapy to achieve the synergistic antitumor effects of both modalities.