Molecular Linking of HIPEC (Hyperthermic Intraperitoneal Chemotherapy) and Tregs (Regulatory T- cells) in Advanced Epithelial Ovarian Cancer - A Review

Ray MD^1*, Deo SVS², Luthra K³, Mathur S⁴, Anand P⁵ and Sharma A^6
*Correspondence: Ray MD, Additional Professor, Department of Surgical Oncology, IRCH, AIIMS, New Delhi-110049, India, Email:

Keywords

Ovarian cancer • HIPEC • Regulatory T-Cells • Immunotherapy • Peritoneal carcinomatosis

Abbreviations

HIPEC: Hyperthermic Intraperitoneal Chemotherapy • FOXP3: Forkhead Box P3 • EOC: Epithelial Ovarian Cancer • Treg: Regulatory T-Cells • CRS: Cytoreductive Surgery • NACT: Neo-Adjuvant Chemotherapy • HSP: Heat Shock Proteins • nTregs: natural Treg cells • iTregs: Inducible Treg Cells • CD: Cluster of Differentiation • CTLA-4: Cytotoxic T-Lymphocyte Antigen 4 • LAG-3: Lymphocyte-Activation Gene 3 • 4-IBB: Belongs to the Tumor Necrosis Factor Receptor Superfamily • IL: Interleukin • NK Cells: Natural Killer Cells • TAA: Tumor-Associated Antigens • APC: Antigen Presenting Cells • TGF: β Transforming Growth Factor • MHC: Major Histocompatibility Complex • RORγ : Retinoic Acid Receptor-Related Orphan Nuclear Receptor Gamma • Th Cells: T-Helper Cells • LTi cells: Lymphoid Tissue Inducer Cells • LBD: Ligand-Binding Domains • ELISA: Enzyme-Linked Immune Sorbent Assay • PB: Peripheral Blood • PF: Peritoneal Fluid • TNF: Tumor Necrosis Factor • IFN: Interferon • TLR: Toll-Like Receptor • HDAC: Histone De-Acetylase • IRCH: Institute Rotary Cancer Hospital • AIIMS: All India Institute of Medical Sciences • ECOG: European Oncology Co-operative Group • RNA: Ribonucleic Acid • cDNA: Complementary DNA • DNA: De-oxy Ribose Nucleic Acid • dNTP: Deoxynucleoside Triphosphates • RT: Reverse Transcriptase • PCR: Polymerase Chain Reaction • BSA: Bovine Serum Albumin • IHC: Immunohistochemistry • ANOVA: Analysis of Variance

Introduction

In advanced Epithelial ovarian cancer (EOC), a subgroup of patients has disease recurrence despite having a good clinical response to platinumbased chemotherapy and complete cytoreductive surgery. A growing number of immune cells have been deciphered over several years. The frequent finding of tumor-associated lymphocytes, as well as differential immunological markers (tumor-associated antigen) from the tumor specimen, provides explicit evidence of immunological response to ovarian cancer. This immune response may have implications with regard to disease prognosis.

In the context of several treatment modalities, ⅔rd of these patients will eventually relapse. The peritoneal cavity is the most common site of relapse. Intraperitoneal therapies have been advocated to command the disease progression. In this context, HIPEC (Hyperthermic intraperitoneal chemotherapy) is one of the effective IP therapies which boosts the therapeutic efficacy by killing tumor cells and inducing an efficient anticancer immune response.

The nomenclature of HIPEC depends on the timing of the intervention in relation to systemic chemotherapy and was described by Mulier et al. It is called as Upfront/ primary CRS and HIPEC -when performed primarily, Interval CRS and HIPEC- when performed after 6 six cycles of NACT, consolidation HIPEC- when done in patients with a complete clinicoradiological response after NACT, secondary CRS and HIPEC-when performed for patients who have a recurrence after CRS or CRS+HIPEC and Salvage CRS + HIPEC-in re-recurrent cases after secondary CRS + HIPEC. However, the fine detail of the molecular mechanisms of HIPEC underlying the association with the immune response is unknown. Here we are endeavoring immense knowledge of immunological aspects of HIPEC in epithelial ovarian cancer by including some evidential fact that HIPEC may be associated with Treg expression.

In this review, we discuss how the significance of HIPEC on genetics and immunology of these patients with cancer, has provided unique insights into the molecular and cellular basis of Treg cells. Studies of HIPEC and its association with Tregs cells should make it possible to increase the paucity of immunotherapeutic modalities of most human cancer at an unprecedented level of molecular and cellular precision. The predictive, preventive, and therapeutic implications of these studies of HIPEC in relation to immunity in EOC may extend to patients with other peritoneal carcinomatoses.

Literature Review

Molecular interaction between HIPEC and Treg

Role of hyperthermia: In HIPEC, heated chemotherapeutic agents are perfused into the peritoneal cavity at 41-43°C with a dwelling time of 45 to 90 min. Although perfusion is increased by hyperthermia in normal tissue, opposite observations were made in tumor tissues with reduced perfusion leading to hypoxia in the tumor cells [1]. Hyperthermia induces the expression of heat shock protein (HSP), thus able to regulate a series of cellular and molecular effects evoking the complex immunological alterations in the host microenvironment. In unstressed cells, HSPs are chaperone proteins that maintain protein configuration and transport. They act as a defense mechanism toward apoptosis-inducing events. The overexpression of HSPs in various tumor entities is associated with poor prognosis. Studies have shown that cytostatic treatment for 1 hour using 5-Fluorouracil, Mitomycin-C, or Oxaliplatin under hyperthermia resulted in significantly increased gene expression of HSPs (especially HSP27, HSP70, and HSP90) compared with normothermic conditions (37°C) as well as corresponding hyperthermic conditions alone without chemotherapy. Even 3 days after exposure, increased HSP expression profiles were observed suggesting similar effects in vivo after a HIPEC procedure [1].

HSPs interact with several survival and anti-apoptotic pathways allowing the cell to deal with potentially lethal conditions. The heat shock proteins provide a defense mechanism by linking up with antigen presentation, crosspresentation, and anti-tumor immunity which all interact with the Regulatory T cells (Tregs) expression (Figure 1). From various studies, it has been proved that Tregs remarkably enhance the immune-suppressive mechanism in the tumor microenvironment of ovarian cancers.

Based on their interference with apoptotic pathways, HSPs have become an interesting target for inhibitory strategies in cancer therapy. Many potential inhibitors of particularly HSP90 have been tested for cancer therapy and currently, HSP90 inhibitors are being evaluated in numerous clinical trials [2]. Next to Ganetespib ( STA-9090), Luminespib (AUY922) in combination with 5-FU and OXA has been described to show a synergistic antiproliferative effect in gastric cancer [2,3]. As HSP90 inhibition has been demonstrated to induce the expression of HSP70, dual targeting of HSP70 and HSP90 might be beneficial to successfully inhibit HSP-dependent antiapoptotic mechanisms [4]. Different small-molecule HSP70 inhibitors such as VER155008 and 2-phenylethynesulfonamide have already been proven to enhance the cytotoxicity of HSP90 inhibitors in oral squamous cell carcinoma [5-7]. For targeting HSP27, 2 small-molecule inhibitors, quercetin and brivudine (RP101) exhibits anti-tumor effects in the prostate, breast, squamous cell, ascites, and gastric cancer cell lines. Similar to AUY922, quercetin potentiates the effects of many first-line chemotherapeutics including doxorubicin, cisplatin, gemcitabine, and 5-FU [8-10]. Interestingly, studies on cell viability data from HSP inhibition assays, using a combination of HSP90 (17-AAG) and HSP70 (VER155008) inhibitors, indeed demonstrated significantly reduced viability of colon cancer cells previously exposed to hyperthermic chemotherapy, suggesting a beneficial effect of HSP inhibition after HIPEC therapy.

Regulatory T-cells: Treg cells can be described as a T-cell population that functionally suppresses an immune response by influencing the activity of another cell type [11,12]. Treg cells were initially described by Gershon et al. in the early 1970s and were called suppressor T cells [13]. Two main groups of Tregs have been characterized based on their site of development i.e., in the thymus (natural Tregs –nTregs) and the periphery (inducible Tregs-iTregs) [14]. NTregs are derived from bone marrow progenitor cells transported to the thymus where they differentiate into nTregs following negative and positive selection. Following maturation, these cells migrate to the periphery [15]. NTregs can be differentiated from other T cells owing to the exclusive expression of forkhead box P3 (FOXP3) which is necessary for nTreg-suppressive function [16,17]. Other essential effector and costimulatory molecules that are expressed by these cells include CD39, CD73, cytotoxic T-lymphocyte antigen 4 (CTLA-4), lymphocyte activation gene 3 (LAG-3), CD28, CD80/86, CD40, OX40 (CD134), and 4-IBB (CD137) [18-20].

iTregs are generated from naive CD4+ T cells subsequent to induction by IL-10 and TGF-β resulting in two populations of iTregs, type 1 Tregs (Tr1), and T helper 3 (Th3) cells, respectively [21-23]. The suppressive function of these cells occurs via IL-10 and TGF-β. Peripheral Tregs can also be generated through interactions between IL-4 or IL-13 and the IL-4Rα. Although FOXP3 is a characteristic marker of nTregs, Th3 cells can also be induced to generate FOXP3 [24-26].

Mechanism of immune modulation by Treg: Tregs suppressive mechanisms transpire through the following mechanisms viz., cytokine secretion, cytolysis, metabolic destruction, and altering of APCs function. The secretion of inhibitory cytokines such as IL-10, TGF-β, and IL-35 suppresses immune function. The inhibitory effects of IL-10 occur via its association with APCs to suppress inflammation hence, in the absence of IL-10-secreting Tregs there is an increase in inflammation [28]. IL-35 on the other hand suppresses the expansion of T cells into other T helper subsets, as well as B cells and macrophages [29,30]. TGF-β is necessary for the survival of Treg subsets [31] (Figure 2). Tregs may suppress the function of other cells via granzyme-mediated killing following the release of granzymes into the target cells [32,33]. Similarly, metabolic disruption involving induction of adenosine and the production of cyclic adenosine monophosphate (cAMP) may be a vital mechanism for suppressing overreactive cells [34]. The versatility in Treg effector function allows them to modulate innate immune cells in particular APCs. This entails the engagement of surface molecules such as CTLA-4 and LAG-3 with MHCII molecules on the APCs conferring inhibitory responses that avert the stimulation of other conventional T cells [35].

FOXP3 gene: FOXP3 (forkhead box P3), also known as scurfin, is a protein involved in immune responses [36]. The gene of FOXP3 is located on the short arm of the X chromosome at Xp.11.233. FOXP3 is a transcription factor that is necessary for the induction of the immunosuppressive functions of regulatory T cells (Treg) (Figure 3). Tregs within the CD4+ CD25+ T cell population are characterized by the expression of the FOXP3+ protein [37,38].

Humans bearing tumors show an elevated amount of Tregs in their blood as well as malignant effusions [39,40]. FOXP3 is reported to be expressed in various kinds of tumor cells including colorectal cancer, melanoma, non-small cell lung cancer, and many other cancer cell lines [41-43]. Sato et al. identified cells in ovarian tumors expressing both CD25 and FOXP3. Recently, the presence of Tregs in ovarian cancer ascites in comparison to normal ascites has been shown [40]. The presence of Tregs in ovarian tumors has been associated with reduced overall survival [44,45]. More specifically, Curiel et al. showed for the first time that CD4+ CD25+ FOXP3+ Treg cells correspond to poor clinical outcomes in epithelial ovarian cancer [44]. The same study also showed that CD4+ CD25+ CD3+ cell populations were much more concentrated in malignant ascites rather than nonmalignant ones and blood. It was also shown that CD4+ CD25+ cells were preferentially concentrated in tumor mass rather than in tumor-draining lymph nodes. Furthermore, the presence of FOXP3 alone was an independent prognostic factor for progressfree and overall survival.

RORγ Th17 Cells: Nuclear hormone receptors (NHRs) form a family of transcription factors that are composed of modular protein structures with DNA- and ligand-binding domains (DBDs and LBDs). The DBDs confer gene target site specificity, whereas LBDs serve as control switches for NHR function.Th17 cells are CD4+ T helper effector cells that express several proinflammatory cytokines including interleukin-17A (IL-17). The NHR RORγt, an immune cell-specific isoform of RORγ (retinoic acid receptor-related orphan nuclear receptor gamma) is a key transcription factor for the development of Th17 cells.

RAR-related orphan nuclear receptor gamma (RORγ) has seen a renewed interest as a potential treatment for a variety of disorders like rheumatoid arthritis, autoimmune disorders, multiple sclerosis, psoriasis, psoriatic arthritis (Figure 3). The pathology of the multiple human autoimmune diseases has shown the involvement of T helper 17 cells and inflammatory cytokines such as IL-17A and IL-17F. Antibodies against IL-17 and IL-23 (Risankizumab) have validated the IL-23 and IL-17 pathway in psoriasis. RORγ exists in two isoforms.

RORγ (also referred to as RORγ1) is expressed in the lung, liver, kidney, muscle, brown fat, and thymus. Furthermore, even though RORγ1 is abundantly expressed, detecting the protein has been challenging. However, RORγt is predominantly highly expressed in the thymus, where it has been identified in immature CD4+/CD8+ thymocytes as well as lymphoid tissue inducer (LTi) cells. The ligand-binding domains (LBDs) of RORγ1 and RORγt are identical, and the crystal structures of the LBD of RORγt with various ligands have been reported.

In a study conducted by Monika Bilska et al. they investigated the potential role of Th17 cells in ovarian cancer patients (n = 71) by analyzing the frequencies of Th17 cells in three different environments, i.e., peripheral blood (PB), peritoneal fluid (PF), and tissue (Th17 infiltrating cells), and the concentration of IL-17A in plasma and PF of patients in terms of their clinical and prognostic significance [46] Th17 cells were analyzed by flow cytometry as a percentage of CD4+ lymphocytes that expressed intracellular expression of IL-17A. The level of IL-17A in plasma and PF were determined by ELISA. Results showed the accumulation of Th17 cells among tumor-infiltrating CD4+ lymphocytes (in relation to PB). Moreover, the percentage of Th17 cells in both PB and PF of OC patients was significantly lower than that in the benign tumors group (n = 35). There were no significant differences in the percentage of Th17 cells in PB, PF, and tissue in relation to clinicopathological characteristics of OC patients and survival. The lower percentage of Th17 cells in the PB and PF of OC patients may promote evasion of host immune response by cancer cells. The concentration of IL-17A in plasma of OC patients was higher than that in both benign tumors and the control group (n = 10). The PF IL-17A level in OC patients was higher than that in women with benign ovarian tumors, indicating its synthesis in the OC microenvironment. Higher IL-17A level in PF is correlated with longer (median: 36.5 vs. 27 months) survival of OC patients.

Interaction between HSP and Treg cells: HSPs are implicated in both the adaptive and innate immune systems. In the innate immune system, HSPs stimulate dendritic cells and macrophages, as these are APCs, they consecutively stimulate adaptive immune cells [47,48]. HSPs are important in NK cell function as they are known to increase cytotoxic function and cell proliferation [49]. In particular, membrane-bound HSP70 on various cancer cells is recognized by cluster of differentiation (CD)-94 on the NK cell, initiating effective cytolysis of the tumor cells [50,51]. HSPs may induce the secretion of either anti- or proinflammatory cytokines thereby monitoring the immune response [52,53].

Similarly, HSPs increase the effectiveness of cross-presentation between antigens and APCs in the extracellular milieu, perpetrating in the presentation of peptides to major histocompatibility complex class one (MHCI) or MHCII molecules on T cells. CD91, an HSP receptor, is a requisite for this process and increases T-cell-mediated responses related to T helper (Th)1, Th2, and Tregs the presence of tumors, concomitant relations between the extracellular HSPs and the APCs following internalization of the tumor peptides via CD91 pathway generate both anti-and proinflammatory immune response mediated by T cells [54,55].

As HSPs regulate an extensive component of the immune system, they likely have a role in the optimal function of most immune cells. Importantly, the chaperoning effects of HSPs are necessary for the induction of certain T-cell phenotypes, importantly, Th1, Th2, and Tregs. This presupposes that HSPs are important in the Treg function. To date, the following HSPs have been investigated in relation to Tregs, HSP60, HSP70, and HSP90. HSPs are important in the induction, proliferation, suppressive function, and cytokine production of Tregs (Figure 4).

HSP60 employs the TLR2-signaling pathway in regulating Treg function. TLR2 is expressed on the surfaces of Tregs [56]. Hence, the association between the TLR2 on the Tregs and the HSP stimulates a sequence of events that affect the functional properties of Tregs. Incidentally, increasing levels of HSP60 have correlated with proportional elevations in the intensity of CD4+ CD25+ Treg-directed suppression on the production of TNF-α and IFN-γ [57]. An increase in HSP60 increases ligand binding of the HSP and the TLR2, thus, increasing suppression. This may represent an autoreactive inflammatory response causing autoimmunity [58]. HSP60 also causes an increase in Treg secretion of TGF-β and IL-10 [57]. HSP60 enhances the differentiation of cord blood cells into CD4+ CD25+ FOXP3+ Tregs [59]. Similarly, costimulatory signals from p277 also increase the activity of CD4+ CD25+ Tregs [57]. Therapeutic administration of HSP60 increases the presence of nTregs, and this is often correlated with a decrease in atherosclerotic plaques, the generation of Tregs, and an increase in the production of TGF-β [60,61]. The concentration of HSP60 affects Treg suppression and proliferation. Hence, with respect to TLR2 on Tregs, strong ligand binding results in Treg proliferation while relatively low levels or interactions of ligands and TLR2 on the Treg result in an increase in Treg suppression [62].

Equally, HSP70 in Tregs promotes heightened suppressive function in Tregs [63]. HSP70 confers its activity via the TLR4 pathway inducing a surge in the regulatory activities of Tregs. The TLR4-signaling pathway is important in Treg function, and this may be important for FOXP3 induction and suppression of inflammatory reactions [64]. TLR4 interactions with HSP70 may also augment effector T cell suppression by Tregs as this has been confirmed in coculture experiments with other ligands [65]. Additionally, the type of Tregs present following HSP administration may be dependent on the type of inflammatory response occurring at the time. For example in the mice model of atherosclerosis, immunization with HSP70 produces a significant amount of CD4+ CD25+ FOXP3+ Tregs [60]. Similarly, adoptive transfer of HSP70 peptide epitopes such as B29 induces antigen-specific FOXP3+ or LAG-3+ CD4+ CD25+.

Tregs that are effective in either aborting or suppressing arthritis in mice [66]. B29 is a highly immunogenic peptide with conserved sequences that are presented to T cells by MHC II molecules. Immunization with HSP70 increases IL-10 producing Tregs [67]. HSP70 derived from Mycobacterium tuberculosis stimulates the proliferation of Tregs by acting through dendritic cells causing a surge in IL-10 while dampening TNF-α release [68]. Additionally, HSP70 has anti-inflammatory properties including down-regulating inflammatory cytokine production, increasing cell and tissue tolerance of cytokine-related cytotoxicity, and influencing the permeability of the epithelial barrier [69].

HSP90 is important for conserving proteins involved in signal transduction, via a multichaperone complex [70]. HSP90 can be regulated by histone deacetylases (HDACs) such as HDAC6, and hypoacetylation of HSP90 occurs in the presence of excessive HDAC6 [71]. HDAC6 belongs to the Class II family of HDACs that are necessary for the removal of acetyl from histones and are found in the nucleus and cytoplasm [72]. In Tregs, the removal of HDAC6 results in the overexpression of HSP90 acetylation resulting in an increase in HSF1-related genes instigating an increase in the suppressive function of Tregs [63]. This may be important in treating patients with colitis. Mice deficient in HDADC6 are more likely to have increased levels of Treg suppression due to the presence of HSP90 and excess FOXP3 [63]. Similarly, mice deficient in HDAC9 have an increased expression of FOXP3 [73]. The presence of HDAC9 has been observed to decrease FOXP3 via deacetylation and incidentally Treg function. HSP70 not only acts via the TLR4 to regulate Tregs but also may inhibit HDAC9 ultimately enhancing the release of Tregs and effective Treg repression [74]. Acetylation is a necessary posttranslational modification process for protein production. Hence, increased acetylation of FOXP3 may avert ubiquitination, increase its regulatory effects, stability, and promote DNA binding [75]. Therapeutic strategies involving the use of HSPs to enhance the availability of FOXP3+ Tregs may be important in autoimmune diseases while in diseases like cancer it may be necessary to inhibit FOXP3 acetylation [76].

Does Treg expression vary with treatment?

In the various laboratory studies, it had been noted that hyperthermia induces HSP in surviving cells. Through its anti-apoptotic properties and upregulation of immunosuppressive FOXP3 Tregs, HSPs contribute to the survival and proliferation of the residual tumor cells. This may look counterproductive and highlights the need for achieving complete cytoreduction (CC- 0- no residual tumor cell and CC-1-residual tumor deposit < 2.5mm). The intriguing point is that these cells were in ideal growth conditions within the nutrient-rich medium. But in patients undergoing HIPEC, the peritonectomy procedure renders the surviving cells hypoxic and heat augments the penetration and cytotoxicity of chemotherapy agents.

We selected patients with advanced epithelial ovarian cancer with FIGO stage III disease undergoing cytoreductive surgery and HIPEC (n=21 with 7 in each group viz., primary, interval, and secondary). The basic clinical and demographic profile was captured. After the Ethical Committee approval, the study was conducted from June 2017 to June 2020. We included patients with a performance status of ECOG 2 and above with epithelial ovarian cancer treated at IRCH AIIMS and willing for regular follow up till 5 years. After written informed consent, samples were taken from the operated specimen (tumor tissue and surrounding normal tissue). The patients were followed up every 3 months for the first two years, then every six months thereafter.

RNA isolation: The freshly collected tissue samples were stored at -80 °C using Trizol. Then about 1ml of Trizol was added to 50-100 mg of tissue. Then 0.2 ml of chloroform was added per 1 ml of trizol and centrifuged at 12,000 g at 4 °C. The aqueous phase was transferred to a new tube and 0.5 ml of isopropanol was added to each ml of the aqueous phase and centrifuged at 12,000 g at 4°C for 10 min. The white RNA pellet was re-suspended in 1 ml of 75% ethanol and centrifuged at 75000 g for 5 min at 4°C. The supernatant was discarded and the pellet was air-dried and re-suspended in 30 mcl of warm RNAse-free water. The RNA purity and yield were checked by nanodrop.

CDNA synthesis: After RNA isolation, cDNA was synthesized using random hexamers. To 1 μl of RNA sample, 1 μl of random hexamers was added with RNAse free water and heated at 65°C for 5 minutes and incubated on ice for 5 minutes Then 1 μl of ribolock, 2 μl of dNTP mix, 1 μl RevertAid RT, and 4 μl of 5x buffer was added.

PCR: To determine the optimum annealing temperatures, a gradient PCR reaction was set up from 55°C to 65°C. To 1 μl of 1: 5 diluted cDNA, 200 μl of dNTP, 1x buffer 1% BSA, 1% PEG, 1U Taq polymerase were added. Initial denaturation was done at 94°C for 2 minutes. This was followed by 35 cycles of PCR. Each cycle includes denaturation at 94°C for 10 seconds; annealing at 55-65°C for 30 seconds; extension at 72°C for 30 seconds and a final extension at 72°C for 5 minutes.

FOXP3: The annealing temperature of 59°C was chosen for FOXP3 because no non-specific bands were observed at this temperature. The exact size of the FOXP3 gene product is 350 bp. Since FOXP3 is GC rich, dimethyl sulfoxide was used in 1.7% concentration to break GC bonds and to reduce the melting temperature of the reaction.

Immunohistochemistry

The formalin-fixed paraffin-embedded tissue blocks were cut at 3-4 micron thickness.IHC for FOXP3 was performed using the Streptavidin Biotin peroxidase technique on the coated section of tumor tissue from all cases using the Invitrogen monoclonal antibody in 1:200 dilution (clone 236A/E7). Tonsil tissue was taken as a positive control (Table 1). IHC for Th17 cells was done using RORγT antibody (source: Biocare clone ACR3208A) in 1: 100 dilution. Tonsil tissue was taken as a positive control.

Discussion

In our study, there was no statistically significant difference in the expression of FOXP3 mRNA between the tumor tissue and the surrounding normal tissue in the upfront group (P=0.1917) and interval group (P=0.7904). However, there was a significant difference observed in the recurrent group (P=0.0039). There was a statistical difference in FOX p3 mRNA expression in the tumor tissue among the three groups. (One way ANOVA F-statistic value=10.87837 and p-value= 0.0008) (Figure 5). This reflects the upregulation of immune-suppressive FOXP3 T regulatory cells in recurrent tumors. There was significant concordance between mRNA and protein expression of FOXP3 Treg cells in tumor tissue. (Spearmann coefficient is 0.84 and 2-tailed P-value=0). It was noted that high levels of FOXP3 Treg cell expression were associated with reduced co-expression of RORγ T-helper cells (Figure 6). FOXP3 Treg is associated with immune suppression-their overexpression helps tumors to evade immune surveillance. RORγ T-helper cells Th17 mediate their actions through interleukin 17A and are associated with autoimmune diseases and are found in Tumor-infiltrating lymphocytes. Reduced expression of RORγ cells helps tumors to evade the immune response.

Conclusion

From this review, we can understand the complex nature of the interaction at a molecular and immunological level in epithelial ovarian cancer patients undergoing HIPEC. Follow-up of these patients with a high level of FOXP3 cells infiltrating the ovarian tumor tissue, especially those with recurrent tumors will help us to correlate with disease-specific survival and the risk of relapse. This could be used to validate FOXP3 tumor-infiltrating lymphocyte expression as a novel and independent prognostic factor in epithelial ovarian cancer (EOC). The results of the change in the proportion of FOXP3 Treg cells in peripheral blood before and after HIPEC is awaited.

Ethics Approval

After the Ethical Committee approval, the study was conducted from June 2017 to June 2020.

Informed Consent

Written informed consent was obtained from the patient for their anonymized information to be published in this article.

Acknowledgments

None.

Funding Information

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Competing Interests

The authors declare that there is no conflict of interest.

Conflict Of Interest

None.

Limitations of the Study

1. Small sample size.

2. Long term follow up is needed for demonstrating survival benefits.

References

1. Grimmig, Tanja, Eva-Maria Moll, Kerstin Kloos And Rebecca Thumm, Et Al. "Upregulated Heat Shock Proteins After Hyperthermic Chemotherapy Point To Induced Cell Survival Mechanisms in Affected Tumor Cells from Peritoneal Carcinomatosis." Cancer Growth Metast 10(2017): 1179064417730559.
2. Tatokoro, Manabu, Fumitaka Koga, Soichiro Yoshida and Kazunori Kihara. "Heat Shock Protein 90 Targeting Therapy: State of the Art and Future Perspective." Excli J 14(2015): 48.
3. Lee, Kyung‐Hun, Ju‐Hee Lee, Sae‐Won HM and Seock‐Ah IM, et al. "Antitumor Activity of NVP‐AUY922, A Novel Heat Shock Protein 90 Inhibitor, in Human Gastric Cancer Cells is Mediated through Proteasomal Degradation of Client Proteins." Cancer Sci 102(2011): 1388-1395.
4. Schilling, Daniela, Carmen Garrido, Stephanie E Combs and Gabriele Multhoff. "The Hsp70 Inhibiting Peptide Aptamer A17 Potentiates Radiosensitization of Tumor Cells by Hsp90 Inhibition." Cancer Letters 390(2017): 146-152.
5. Davenport, EL, A Zeisig, LI Aronson and HE Moore, et al. "Targeting Heat Shock Protein 72 Enhances Hsp90 Inhibitor-Induced Apoptosis in Myeloma." Leukemia 24(2010): 1804-1807.
6. Massey, Andrew J, Douglas S Williamson, Helen Browne and James B Murray, et al. "A Novel, Small Molecule Inhibitor of Hsc70/Hsp70 Potentiates Hsp90 Inhibitor Induced Apoptosis in HCT116 Colon Carcinoma Cells." Cancer Chemother Pharmacol 66(2010): 535-545.
7. Wang, Y and SR Mcalpine. "Combining an Hsp70 Inhibitor with either an N-Or C-Terminal Hsp90 Inhibitor Produces Mechanistically Distinct Phenotypes." Organic Biomol Chem 13(2015): 3691-3698.
8. Mcconnell, Jeanette R and Shelli R Mcalpine. "Heat Shock Proteins 27, 40, And 70 As Combinational and Dual Therapeutic Cancer Targets." Bioorg Med Chem Letters 23(2013): 1923-1928.
9. Staedler, Davide, Elita Idrizi, Blanka Halamoda Kenzaoui and Lucienne Juillerat-Jeanneret. "Drug Combinations with Quercetin: Doxorubicin plus Quercetin in Human Breast Cancer Cells." Cancer Chemother Pharmacol 68(2011): 1161-1172.
10. Xavier, Cristina PR, Cristovao F Lima, Mikkel Rohde and Cristina Pereira-Wilson. "Quercetin Enhances 5-Fluorouracil-Induced Apoptosis in MSI Colorectal Cancer Cells through P53 Modulation." Cancer Chemother Pharmacol 68(2011): 1449-1457.
11. Shevach, Ethan M. "Certified Professionalscd4+ CD25+ Suppressor T Cells." J Exp Med 193(2001): F41-F46.
12. Mudd, PA, BN Teague and AD Farris. "Regulatory T Cells and Systemic Lupus Erythematosus." Scandinavian J Immunol 64(2006): 211-218.
13. Gershon, Richard K, Philip Cohen, Ronald Hencin and Stephen A Liebhaber. "Suppressor T Cells." J Immunol 108(1972): 586-590.
14. Workman, CJ, AL Szymczak-Workman, LW Collison and MR Pillai, et al. “The Development and Function of Regulatory T Cells.” Cellular Mol Life Sci 66(2009): 2603-2622.
15. Shevach, Ethan M. "CD4+ CD25+ Suppressor T Cells: More Questions than Answers." Nature Rev Immunol 2(2002): 389-400.
16. Sakaguchi, S. “Naturally Arising FOXP3-Expressing CD25+CD4+ Regulatory T Cells in Immunological Tolerance to Self and Non-Self.” Nat Immunol 6(2005): 345-352.
17. Sakaguchi, Shimon. "Naturally Arising CD4+ Regulatory T Cells for Immunologic Self-Tolerance and Negative Control of Immune Responses." Annu Rev Immunol 22(2004): 531-562.
18. Huang, Shuo, Lei Li, Songlan Liang and Weizhi Wang. "Conversion Of Peripheral CD4+ CD25− T Cells To CD4+ CD25+ Regulatory T Cells by IFN-Γ in Patients with Guillain-Barré Syndrome." J Neuroimmunol 217(2009): 80-84.
19. Chen, Wanjun, Wenwen Jin, Neil Hardegen and Ke-Jian Lei, et al.  "Conversion of Peripheral CD4+ CD25− Naive T Cells To CD4+ CD25+ Regulatory T Cells by TGF-Β Induction of Transcription Factor FOXP3." J Exp Med 198(2003): 1875-1886.
20. Kretschmer, Karsten, Irina Apostolou, Daniel Hawiger and Khashayarsha Khazaie, et al.  "Inducing and Expanding Regulatory T Cell Populations by Foreign Antigen." Nat Immunol 6(2005): 1219-1227.
21. Kaplan, Mark H, Ya-Lin Sun, Timothy Hoey and Michael J Grusby. "Impaired IL-12 Responses and Enhanced Development of Th2 Cells in Stat4-Deficient Mice." Nature 382(1996): 174-177.
22. Seder, Robert A, Ricardo Gazzinelli, Alan Sher and William E Paul. "Interleukin 12 Acts Directly on CD4+ T Cells to Enhance Priming for Interferon Gamma Production and Diminishes Interleukin 4 Inhibition of Such Priming." Proc Nat Acad Sci 901993): 10188-10192.
23. Cottrez, Françoise and Hervé Groux. "Specialization in Tolerance: Innate CD4+ CD25+ Versus Acquired TR1 and TH3 Regulatory T Cells. 1." Transplant 77(2004): S12-S15.
24. Carrier, Yijun, Jing Yuan, Vijay K Kuchroo and Howard L Weiner. "Th3 Cells in Peripheral Tolerance. I. Induction Of FOXP3-Positive Regulatory T Cells by Th3 Cells Derived from TGF-Β T Cell-Transgenic Mice." J Immunol 178(2007): 179-185.
25. Maggi, Enrico, Lorenzo Cosmi, Francesco Liotta and Paola Romagnani, et al. "Thymic Regulatory T Cells." Autoimmun Rev 4(2005): 579-586.
26. Taams, LS and AN Akbar, “Peripheral Generation and Function of CD4+CD25+ Regulatory T Cells.” Curr Topics Microbiol Immunol 293(2005): 115-131.
27. Shevach, Ethan M. "Mechanisms of FOXP3+ T Regulatory Cell-Mediated Suppression." Immunity 30(2009): 636-645.
28. Sojka, Dorothy K, Yu‐Hui Huang and Deborah J Fowell. "Mechanisms of Regulatory T‐Cell Suppression–A Diverse Arsenal for a Moving Target." Immunol 124(2008): 13-22.
29. Whitehead, Gregory S, Rhonda H Wilson, Keiko Nakano And Lauranell H Burch, et al. "IL-35 Production by Inducible Costimulator (ICOS)–Positive Regulatory T Cells Reverses Established IL-17–Dependent Allergic Airways Disease." J Allergy Clin Immunol 129(2012): 207-215.
30. Collison, Lauren W, Creg J Workman, Timothy T Kuo and Kelli Boyd, et al. "The Inhibitory Cytokine IL-35 Contributes to Regulatory T-Cell Function." Nature 450(2007): 566-569.
31. Yamagiwa, Satoshi, J Dixon Gray, Shigeo Hashimoto and David A Horwitz. "A Role for TGF-Β in the Generation and Expansion of CD4+ CD25+ Regulatory T Cells from Human Peripheral Blood." J Immunol 166(2001): 7282-7289.
32. Czystowska, Malgorzata, Laura Strauss, Christoph Bergmann and Marta Szajnik, et al. "Reciprocal Granzyme/Perforin-Mediated Death of Human Regulatory and Responder T Cells Is Regulated By Interleukin-2 (IL-2)." J Mol Med 88(2010): 577-588.
33. Cao, Xuefang, Sheng F Cai, Todd A Fehniger And Jiling Song, et al.  "Granzyme B And Perforin Are Important for Regulatory T Cell-Mediated Suppression of Tumor Clearance." Immunity 27(2007): 635-646.
34. Vaeth, Martin, Tea Gogishvili, Tobias Bopp and Matthias Klein, et al. "Regulatory T Cells Facilitate the Nuclear Accumulation of Inducible Camp Early Repressor (ICER) and Suppress Nuclear Factor of Activated T Cell C1 (Nfatc1)." Proc Nat Acad Sci 108(2011): 2480-2485.
35. Zhu, Xuefei, Peizeng Yang, Hongyan Zhou and Bing Li, et al. "CD4+ CD25+ Tregs Express an Increased LAG-3 and CTLA-4 in Anterior Chamber-Associated Immune Deviation." Graefe's Arch Clin Exp Ophthalmol 245(2007): 1549-1557.
36. Brunkow, Mary E, Eric W Jeffery, Kathryn A Hjerrild And Bryan Paeper, et al. "Disruption of a New Forkhead/Winged-Helix Protein, Scurfin, Results in the Fatal Lymphoproliferative Disorder of the Scurfy Mouse." Nat Genet 27(2001): 68-73.
37. Sato, Eiichi, Sara H Olson, Jiyoung Ahn and Brian Bundy, et al. "Intraepithelial CD8+ Tumor-Infiltrating Lymphocytes and a High CD8+/Regulatory T Cell Ratio are Associated with Favorable Prognosis in Ovarian Cancer." Proc Nat Acad Sci 102(2005): 18538-18543.
38. Kryczek, Ilona, Rebecca Liu, Guobin Wang and Ke Wu, et al.  "FOXP3 Defines Regulatory T Cells in Human Tumor and Autoimmune Disease." Cancer Res 69(2009): 3995-4000.
39. Woo, Edward Y, Christina S Chu, Theresa J Goletz and Katia Schlienger, et al. "Regulatory CD4+ CD25+ T Cells In Tumors from Patients with Early-Stage Non-Small Cell Lung Cancer and Late-Stage Ovarian Cancer." Cancer Res 61(2001): 4766-4772.
40. Bamias, A, ML Tsiatas, E Kafantari And C Liakou, et al. "Significant Differences Of Lymphocytes Isolated from Ascites of Patients with Ovarian Cancer Compared to Blood and Tumor Lymphocytes. Association of CD3+ CD56+ Cells with Platinum Resistance." Gynecologic Oncol 106(2007): 75-81.
41. Schultze, Frank Christian, Reiner Andag, Salamah Mohammad Alwahsh and Draga Toncheva, et al. "FOXP3 Demethylation is increased in Human Colorectal Cancer and Rat Cholangiocarcinoma Tissue." Clin Biochem 47(2014): 201-205.
42. Tan, Beeshin, Matthew Anaka, Siddhartha Deb and Claudia Freyer, et al. "FOXP3 Over-Expression Inhibits Melanoma Tumorigenesis via Effects on Proliferation and Apoptosis." Oncotarget 5(2014): 264.
43. Fu, Hai-Ying, Chun Li, Wei Yang and Xiao-Dong Gai, et al. "FOXP3 and TLR4 Protein Expression are correlated in Non-Small Cell Lung Cancer: Implications for Tumor Progression and Escape." Acta Histochemica 115(2013): 151-157.
44. Curiel, Tyler J, George Coukos, Linhua Zou and Xavier Alvarez, et al. "Specific Recruitment of Regulatory T Cells in Ovarian Carcinoma Fosters Immune Privilege and Predicts Reduced Survival." Nat Med 10(2004): 942-949.
45. Wolf, Dominik, Anna M Wolf, Holger Rumpold and Heidi Fiegl, et al. "The Expression of the Regulatory T Cell–Specific Forkhead Box Transcription Factor FOXP3 is Associated with Poor Prognosis in Ovarian Cancer." Clin Cancer Res 11(2005): 8326-8331.
46. Bilska, Monika, Anna Pawłowska, Ewelina Zakrzewska and Agata Chudzik, et al. "Th17 Cells and IL-17 as Novel Immune Targets in Ovarian Cancer Therapy." J Oncol 12(2020): 2-4.
47. Facciponte, John G, Ian J Macdonald, Xiang-Yang Wang and Hyung Kim, et al. "Heat Shock Proteins and Scavenger Receptors: Role in Adaptive Immune Responses." Immunol Investig 34(2005): 325-342.
48. Kumaraguru, Udayasankar, Christopher D Pack and Barry T Rouse. "Toll‐Like Receptor Ligand Links Innate And Adaptive Immune Responses by the Production of Heat‐Shock Proteins." J Leukocyte Biol 73(2003): 574-583.
49. Multhoff, Gabriele. "Activation of Natural Killer Cells by Heat Shock Protein 70." Int J Hyperthermia 18(2002): 576-585.
50. Gross, Catharina, Ingo GH Schmidt-Wolf, Srinivas Nagaraj and Robert Gastpar, et al. "Heat Shock Protein 70-Reactivity is Associated with Increased Cell Surface Density of CD94/CD56 on Primary Natural Killer Cells." Cell Stress Chaperones 8(2003): 348.
51. Stangl, Stefan, Catharina Gross, Alan G Pockley and Alexzander A Asea, et al. "Influence Of Hsp70 and HLA-E on the Killing of Leukemic Blasts by Cytokine/Hsp70 Peptide-Activated Human Natural Killer (NK) Cells." Cell Stress Chaperones 13(2008): 221-230.
52. Tsan, Min‐Fu and Baochong Gao. "Heat Shock Proteins and Immune System." J Leukocyte Biol 85(2009): 905-910.
53. Van Eden, Willem, Ruurd Van Der Zee and Berent Prakken. "Heat-Shock Proteins Induce T-Cell Regulation of Chronic Inflammation." Nat Rev Immunol 5(2005): 318-330.
54. Calderwood, Stuart K, Salamatu S Mambula and Phillip J Gray. "Extracellular Heat Shock Proteins in Cell Signaling and Immunity." Ann New York Acad Sci 1113(2007): 28-39.
55. Pawaria, Sudesh, Michelle Nicole Messmer, Yu Jerry Zhou and Robert Julian Binder. "A Role for the Heat Shock Protein–CD91 Axis in the Initiation of Immune Responses to Tumors." Immunol Res 50(2011): 255-260.
56. Liu, Haiying, Mousa Komai-Koma, Damo Xu and Foo Y Liew. "Toll-Like Receptor 2 Signaling Modulates the Functions Of CD4+ CD25+ Regulatory T Cells." Proc Nat Acad Sci 103(2006): 7048-7053.
57. Zanin-Zhorov, Alexandra, Liora Cahalon, Guy Tal and Raanan Margalit, et al. "Heat Shock Protein 60 Enhances CD4+ CD25+ Regulatory T Cell Function via Innate TLR2 Signaling." J Clin Investig 116(2006): 2022-2032.
58. Sakaguchi M, Kitahara H, Seto T. "Surgery for Acute Type a Aortic Dissection In Pregnant Patients with Marfan Syndrome.” Eur J Cardiothorac Surg 28(2005): 280-283.
59. Aalberse, Joost A, Berber Kapitein, Sytze De Roock and Mark R Klein, et al. "Cord Blood CD4+ T Cells Respond to Self-Heat Shock Protein 60 (HSP60)." Plos One 6(2011): E24119.
60. Van Puijvelde, Gijs HM, Thomas Van Es and EJA Van Wanrooij, et al. "Induction of Oral Tolerance to HSP60 or an HSP60-Peptide Activates T Cell Regulation and Reduces Atherosclerosis." Arterioscl Thromb Vasc Biol 27(2007): 2677-2683.
61. Li, Haiyu, Yanping Ding, Guiwen Yi and Qiutang Zeng, et al. "Establishment of Nasal Tolerance to Heat Shock Protein-60 Alleviates Atherosclerosis by Inducing TGF-Β-Dependent Regulatory T Cells." J Huazhong Univ Sci Technol 32(2012): 24-30.
62. Sutmuller, Roger, Anja Garritsen and Gosse J Adema. "Regulatory T Cells and Toll-Like Receptors: Regulating the Regulators." Ann Rheu Dis 66(2007): 91-95.
63. De Zoeten, Edwin F, Liqing Wang, Kyle Butler and Ulf H Beier, et al. "Histone Deacetylase 6 and Heat Shock Protein 90 Control the Functions of FOXP3+ T-Regulatory Cells." Mol Cellul Biol 31(2011): 2066-2078.
64. Dai, Jie, Bei Liu, Soo Mun Ngoi and Shaoli Sun, et al. "TLR4 Hyperresponsiveness via Cell Surface Expression of Heat Shock Protein Gp96 Potentiates Suppressive Function of Regulatory T Cells." J Immunol 178(2007): 3219-3225.
65. Caramalho, Iris, Thiago Lopes-Carvalho, Dominique Ostler and Santiago Zelenay, et al. "Regulatory T Cells Selectively Express Toll-Like Receptors and are Activated by Lipopolysaccharide." J Exp Med 197(2003): 403-411.
66. Van Herwijnen, Martijn JC, Lotte Wieten and Ruurd Van Der Zee, et al. "Regulatory T Cells that Recognize a Ubiquitous Stress-Inducible Self-Antigen are Long-Lived Suppressors of Autoimmune Arthritis." Proc Nat Acad Sci 109(2012): 14134-14139.
67. Wieten, Lotte, Suzanne E Berlo, B Corlinda and Peter J Van Kooten, et al. "IL-10 Is Critically Involved In Mycobacterial HSP70 Induced Suppression of Proteoglycan-Induced Arthritis." PLOS One 4(2009): E4186.
68. Borges, Thiago J, Bárbara N Porto, César A Teixeira and Marcelle Rodrigues, et al. "Prolonged Survival of Allografts Induced by Mycobacterial Hsp70 is Dependent on CD4+ CD25+ Regulatory T Cells." Plos One 5(2010): E14264.
69. Jäättelä, Marja, Dorte Wissing, Klaus Kokholm and Tuula Kallunki, et al. "Hsp70 Exerts Its Anti‐Apoptotic Function Downstream of Caspase‐3‐Like Proteases." EMBO J 17(1998): 6124-6134.
70. Young, Jason C, Ismail Moarefi and F Ulrich Hartl. "Hsp90: A Specialized but Essential Protein-Folding Tool." J Cell Biol 154(2001): 267-274.
71. Aoyagi, Sayura and Trevor K Archer. "Modulating Molecular Chaperone Hsp90 Functions through Reversible Acetylation." Trends Cell Biol 15(2005): 565-567.
72. Zhang, Hongtao, Yan Xiao, Zhiqiang Zhu and Bin Li, et al. "Immune Regulation by Histone Deacetylases: A Focus on the Alteration of FOXP3 Activity." Immunol Cell Biol 90(2012): 95-100.
73. Tao R, De Zoeten EF and Ozkaynak E. "Deacetylase Inhibition Promotes the Generation and Function of Regulatory T Cells." Nat Med 13(2007): 1299-1307.
74. De Zoeten, Edwin F, Liqing Wang, Hong Sai and Wolfgang H Dillmann, et al. "Inhibition of HDAC9 Increases T Regulatory Cell Function and Prevents Colitis in Mice." Gastroenterol 138(2010): 583-594.
75. Xiao, Yan, Bin Li, Zhaocai Zhou and Wayne W Hancock, et al. "Histone Acetyltransferase Mediated Regulation of FOXP3 Acetylation and Treg Function." Curr Opinion Immunol 22(2010): 583-591.
76. Beier, Ulf H, Tatiana Akimova, Yujie Liu and Liqing Wang, et al. "Histone/Protein Deacetylases Control FOXP3 Expression and the Heat Shock Response of T-Regulatory Cells." Curr Opinion Immunol 23(2011): 670-678.