Ovarian Cancer: Stem cell treatment to the rescue

Ovarian cancer is the fifth most common cause of cancer among women, accounting for more deaths than any other cancer of the female reproductive system. A woman’s risk of getting ovarian cancer during her lifetime is about 1 in 78.

Due to later stages of diagnosis and reduced therapeutic efficacy, ovarian cancer is one of the most fatal gynaecological malignancies. Treatments for this cancer have changed little over the past few decades, with surgery and chemotherapy being the most common therapeutic approaches. Although rapid improvements occur in multiple therapy strategies, the clinical outcome has not been improved in ovarian cancer patients. Thus, innovative therapeutic strategies are needed to achieve successful management of ovarian cancer. Consequently, new treatment strategies such as immunotherapy are appearing. Numerous investigations are underway on immune checkpoint blockade, cancer vaccines, and adoptive cell therapy immune therapies for ovarian cancer.

Immunotherapy, a type of treatment that activates a patient’s immune system to target cancer cells, has been successful in many diseases but not ovarian cancer and it is unclear why. Adoptive cell therapy is a rapidly expanding approach to cancer immunotherapy to facilitate antitumour reactions by introducing potent effector cells into the tumour microenvironment. In this context, neutrophils, and natural killer cells as a substantial proportion of the innate immune system have been reported to exert anti-tumourigenic roles in the tumour microenvironment.

How do Neutrophils and Natural Killer Cells work?

Neutrophils are the most abundant circulating leukocytes, accounting for 50–70% of blood cells. During neutrophil maturation from common myeloid progenitors, the granulocyte-monocyte progenitor cells are the first to acquire the neutrophil lineage marker CD66b. Then, during maturation, from promyelocytes to human mature neutrophils, the simultaneous acquisition of CD11b and CD16 can be observed (Figure 1).

Human neutrophils (A) are characterized by CD11b, polarization, neutrophils in the tumour microenvironment are also characterized by the capability to acquire two opposite phenotypes. N1 neutrophils (B) show antitumoural activities and are characterized by the expression
of chemokine (C-C motif) ligand 3 (CCL3), tumour necrosis factor α (TNFα), arginase, and intercellular adhesion molecule 1 (ICAM1); in contrast, N2 neutrophils (C) work as tumour-promoters and express CC and CXC chemokines (CCL2, 3, 4, 8, 12 and 17 and CXCL1, 2, 8, and 16), vascular endothelial growth factor (VEGF), matrix metalloprotease 9 (MMP9), and CXCR4 receptor. Human NK cell subsets (D) are characterized by CD56 expression. Cytolytic NK cells express CD56 and CD16 (CD56dimCD16+) and can release perforin and granzyme; cytokine-producer NK cells lose CD16 expression and increase CD56 expression (CD56brigthCD16−) with the production of cytokines including TNFα and interferon γ (IFNγ); the last subset is named decidual cells (dNKs) that displayed higher expression of CD56 with CD9 and CD49a markers (CD56superbrigthCD16−CD9+CD49a+) and support angiogenesis through the release of VEGF, placental growth factor (PlGF), and interleukin 6 (IL6). A similar subset named decidual-like NK cells is also found in the tumour microenvironment (TME).

Neutrophils represent the first-line defence against infections, thus they are rapidly recruited from the bloodstream to the site of injury where they eliminate pathogens, particularly bacteria, by phagocytosis, degranulation, and release of extracellular traps (NETs).

In both macrophages and neutrophils opposite functions or heterogeneity has been described, and the existence of opposing polarization states (pro-tumoural or anti-tumoural) of macrophages and neutrophils in cancer. In cancer, mimicking the dichotomy between proinflammatory/ M1-like and anti-inflammatory/tissue-repairing/TAM/M2-like macrophages, neutrophils have been conventionally termed N1 and N2 neutrophils, with the latter characterized by immunosuppressive, pro-metastatic, and pro-angiogenic activities.

Natural killer (NK) cells are large granular lymphocytes from innate immunity, participating in the early recognition and elimination of virus-infected and malignant-transformed cells. Currently, NK cells are classified as innate lymphoid cells (ILCs) and originate from common innate lymphoid progenitors in the bone marrow. Subsequently, they migrate to different lymphoid tissues or non-lymphoid tissues, where they are “educated”, acquiring phenotype and functions typical of tissue residency. The NK inhibitory receptor repertoire is adapted to the MHC class I molecules borne by the host, assuring NK cell tolerance against self-cells. On the other hand, NK cells are simultaneously stimulated by activating receptors that trigger NK cell responses. In the presence of healthy cells, activating signals are low, thus the binding of inhibitory receptors to MHC class I molecules is sufficient to induce NK cell tolerance. In contrast, when NK cells recognize altered cells (i.e., tumour cells) that lack or downregulate MHC I expression, the activating signals overcome the inhibitory ones, leading to the killing of altered cells.

Neutrophils can (A) induce a reduction in CCR1 expression in NK cells, impairing the NK cells’ infiltration capability. Interference with PDL1-PD1 (B) interactions in the TME, resulting in reduced NK cell capability to release IFNγ. Neutrophils can also modulate the expression of activating receptor NKp46 on NK cells through (C) the release of different molecules that include: (i) neutrophil-derived Cathepsin G (CG) reduces NKp46 on NK cells; similar; (ii) reactive oxygen species (ROS) can downmodulate NKp46 on cytotoxic CD56dimCD16+NK cells while they can upregulate this receptor on cytokine-producer CD56brigthCD16− NK cells; (iii) elastase and lactoferrin release exerts a wide effect increasing cytotoxicity and reducing angiogenesis. NK-derived IFNγ, a key mediator in TME, can be inversely modulated by ARG1 and IL12 from neutrophils (D). (i) Indeed, neutrophil-derived ARG1 can abrogate IFNγ released from NK, improving NK pro-angiogenic features; (ii) while neutrophil-derived IL12 through STAT4 activation increases IFNγ and perforin production by NK cells. NK cell–neutrophil crosstalk (E) can be modulated by NK-derived IFNγ, which acts by decreasing pro-angiogenic features of tumour-associated neutrophils (TANs). Indeed, NK cells or IFNγ depletion increase TANs’ pro-angiogenic features. (1)

Tumour-associated neutrophils have been shown to robustly produce a variety of toxic
compounds and can induce tumour cell cytolysis or cytostasis in vitro, which suggests that
in certain circumstances they might oppose tumourigenesis. The mechanisms of neutrophils
killing cancer cells may be direct cytotoxicity toward tumour cells. Neutrophils can be polarized
into different phenotypes according to various cytokines secreted by cancer cells in the tumour
microenvironment.

Focus on Immunotherapy, mesenchymal stem cells and stem cell secreted factors

The current focus of research on ovarian is to improve our understanding of the immune environment in ovarian cancer in hopes of making immunotherapy an option for these patients. Recent evidence has pointed to key characteristics of immune cells in ovarian cancer and
identified cell types important for mediating an immune response.

To obtain an adoptive cell therapy (ACT) exploiting allogeneic neutrophils which are easily available and abundant; umbilical cord blood-derived neutrophils offer superior amounts over other sources of neutrophils. For example, researchers have shown that LPS and IL-8-activated neutrophils from umbilical cord blood (UCB) could inhibit the progression of ovarian cancer cells. Thus, providing strong evidence of UCB-derived neutrophil-based immunotherapy against ovarian cancer (2-3).

Various ovarian cancer xenograft models have been established to evaluate the efficacy of NK cells. Data to support this is provided; for example, SR1/IL–15/IL12 expanded UCB-NK cells constitute a promising immunotherapeutic product that can be exploited for intraperitoneal therapy of ovarian cancer patients, as demonstrated by their capability to actively migrate, infiltrate, and mediate intra-tumoural cell killing in OC spheroids. In addition, promising preclinical anti-ovarian cancer activity of UCB-NK cells was also demonstrated following intraperitoneal infusion in a relevant SKOV-3-based ovarian cancer xenograft model (4-5).

Premature ovarian insufficiency (POI) and infertility are common and severe side effects of chemotherapy. Chemotherapeutic agents target oocytes directly or induce oocyte death indirectly by damaging somatic cells.

Recently, stem cell transplantation has been shown to be a new strategy for the treatment of POI and infertility following chemotherapy. Based on the evidence, stem cells enhance ovarian function due to their paracrine effects rather than differentiating into specific cells. Studies on stem cell-derived secreted factors show that the secretome, microvesicles, and exosome are all found in the medium where stem cells were cultured, which is known as conditioned medium (CM). The contents of these vesicles secreted by mesenchymal stem cells (MSCs) include cytokines and growth factors, signalling lipids, messenger RNAs (mRNAs), and regulatory miRNAs. These factors are involved in cell–cell communication, cell signalling, and alteration of cell or tissue metabolism. CM could promote tissue/organ repair under various conditions. In addition, CM has several advantages compared with stem cells. (i) CM can be manufactured, freeze-dried, packaged, and transported more easily, (ii) it eliminates issues with matching the donor and the recipient to avoid rejection problems; and (iii) it eliminates the potential side effects of stem cells on tumour cells, such as differentiating into other stromal cell types, increasing the metastatic abilities of tumour cells, and stimulating the epithelial–mesenchymal transition of tumour cells.

In addition, recent studies showed that Human umbilical cord mesenchymal stem cell (hUCMSC) transplantation can reduce cumulus cell apoptosis, and restore ovarian function. Even primordial follicles in the ovaries treated with hUCMSC-CM exhibited an apparent resistance to cisplatin. Additionally, ovarian reserve and fertility can also be preserved after hUCMSC-CM treatment. CM derived from human amniotic epithelial cells (hAECs) can protect ovaries against chemotherapy-induced damage and 109 cytokines in hAEC-CM might participate in apoptosis, angiogenesis, cell cycle, and immune response.

In conclusion, the value of hUCMSCs to exert protective effects on cisplatin-induced ovarian damage via the paracrine pathway has clearly been demonstrated. These results will add value in promoting the application of CM in clinical treatment, and hopefully, infertile patients can benefit from hUCMSC-CM treatment in the future (7-18).

All the above results demonstrate that ovarian cancer, despite resistance to existing immunotherapies, is indeed an immunogenic disease and provide a roadmap for the design of improved immunotherapy options by using allogeneic stem cell-based therapies, which could even be applicable to other tumours with similar mutational burden.

In addition, MSCs cells are easily accessible, do not induce immunological responses, can be simply manipulated in vitro without the requirement for immortalization; can preferentially migrate into the tumour tissues and have transactions with various cells in the tumour microenvironment. MSCs therefore, are the most appropriate choices for cell-based treatments in cancers. Notwithstanding, the progress in obtaining enough autologous and allogeneic MSCs from bone marrow, adipose tissue, umbilical cord blood, and local tissues, however, their mechanisms of action still require further investigation. Additionally, exosomes released by MSCs and their manipulation may offer another promising technique in MSC-based cancer therapy. Despite providing a novel and attractive therapeutic system in cancer therapy, MSCs have been accompanied by several challenging issues and limitations.

Thus, future clinical trials to evaluate the efficacy of immunotherapy and MSC-based treatment strategies in ovarian cancer might prove imperative in the battle against this fatal gynaecological malignancy.

References

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