Comparing the Secretomes of Chemorefractory and Chemoresistant Ovarian Cancer Cell Populations

Simple Summary Epithelial ovarian cancer (EOC) is a gynecological disease that is complicated to treat due to its heterogenous nature and because many women develop resistance to various therapeutic strategies. Tumor recurrence can be examined as a two-pronged approach: resistance developed after multiple exposures to frontline anticancer drugs or resistance developed in response to poor microenvironmental conditions, such as hypoxia. Although there are numerous ways to confer chemoresistance, studies have shown that chemoresistant EOC cells release unique secretome profiles that include cytokines, growth factors, and extracellular vesicles (EVs). These secreted factors activate intracellular pathways that contribute to chemoresistance. Secreted EVs transfer biomaterials (including proteins, RNAs, and microRNAs) to other cells, which is critical in cell–cell communication; thus, changes in EV content, in particular exosome miRNAs, have been used to project EOC prognosis. This review examines the feedback loop where chemoresistant EOC cells release unique secretome profiles that confer chemoresistance in normal bystander cells and cancer cells. Abstract High-grade serous ovarian cancer (HGSOC) constitutes the majority of all ovarian cancer cases and has staggering rates of both refractory and recurrent disease. While most patients respond to the initial treatment with paclitaxel and platinum-based drugs, up to 25% do not, and of the remaining that do, 75% experience disease recurrence within the subsequent two years. Intrinsic resistance in refractory cases is driven by environmental stressors like tumor hypoxia which alter the tumor microenvironment to promote cancer progression and resistance to anticancer drugs. Recurrent disease describes the acquisition of chemoresistance whereby cancer cells survive the initial exposure to chemotherapy and develop adaptations to enhance their chances of surviving subsequent treatments. Of the environmental stressors cancer cells endure, exposure to hypoxia has been identified as a potent trigger and priming agent for the development of chemoresistance. Both in the presence of the stress of hypoxia or the therapeutic stress of chemotherapy, cancer cells manage to cope and develop adaptations which prime populations to survive in future stress. One adaptation is the modification in the secretome. Chemoresistance is associated with translational reprogramming for increased protein synthesis, ribosome biogenesis, and vesicle trafficking. This leads to increased production of soluble proteins and extracellular vesicles (EVs) involved in autocrine and paracrine signaling processes. Numerous studies have demonstrated that these factors are largely altered between the secretomes of chemosensitive and chemoresistant patients. Such factors include cytokines, growth factors, EVs, and EV-encapsulated microRNAs (miRNAs), which serve to induce invasive molecular, biophysical, and chemoresistant phenotypes in neighboring normal and cancer cells. This review examines the modifications in the secretome of distinct chemoresistant ovarian cancer cell populations and specific secreted factors, which may serve as candidate biomarkers for aggressive and chemoresistant cancers.

Introduction
Ovarian cancers are broadly categorized based on the tissue of origin into three groups-epithelial, germ cell, and stromal cell [1]. Epithelial ovarian cancer (EOC) is the predominant diagnosis, accounting for 90% of new ovarian cancer cases, and often originates from the fallopian tubes [2]. High-grade serous ovarian cancer (HGSOC) accounts for 75% of all EOC cases, making HGSOC the predominant form of ovarian cancer. HGSOC has a mortality rate of 60% and is often diagnosed at advanced stages, post-metastasis [1]. A major contributing factor to the high mortality rate is that up to 25% of patients experience refractory disease and are inherently resistant to frontline treatment. Furthermore, of the remaining patients that initially respond to treatment, 70% acquire resistance and experience disease recurrence [1,3]. These statistics suggest that refractory and resistant HGSOC are all too common and highlight our gap in understanding how resistance is developed prior to, and as a result of, chemotherapeutic intervention.
Chemoresistance can be classified as either intrinsic or acquired, based on when the resistance is developed. Prior to chemotherapy, refractory cancers develop an intrinsic resistance to anticancer drugs [4] which demonstrates the priming and protective capabilities of the tumor microenvironment (TME). The HGSOC TME is comprised of a host of stromal cells recruited by cancer cells (e.g., fibroblasts, immune cells, endothelial cells) and the extracellular matrix (ECM) developed and maintained by the constant bidirectional communication between the cancer and stromal cell populations. The TME can be protective and protumorigenic; however, as a consequence of a rapidly growing cancer, the TME can also be a hostile environment with limitations in oxygen diffusion, nutrient depletion, and increased mechanical pressure. Each of these stressors can be cytotoxic to cancer cells [5][6][7]; and yet, under the right circumstances, exposure to such stress can serve as a potent trigger to dramatically modify and prime the TME for future insults ( Figure  1).

Figure 1.
Both hypoxia in the tumor microenvironment and chemotherapy confer resistance to stress and, ultimately, chemotherapies. Each drives distinct changes in the TME composition in terms of cell populations and the secretome. Modifications in the HGSOC secretome as a result of TME stressors not only promote the development of chemotherapy-induced resistance, but are also expanded after exposure to anticancer drugs, thus reinforcing a protective and resilient TME. Abbreviations: (p)EMT-(partial) epithelial-to-mesenchymal transition; CSC-cancer stem cell; HGSOChigh-grade serous ovarian cancer; SASP-senescence-associated secretory phenotype; EV-extracellular vesicles (generated with BioRender, Toronto, Canada). Both hypoxia in the tumor microenvironment and chemotherapy confer resistance to stress and, ultimately, chemotherapies. Each drives distinct changes in the TME composition in terms of cell populations and the secretome. Modifications in the HGSOC secretome as a result of TME stressors not only promote the development of chemotherapy-induced resistance, but are also expanded after exposure to anticancer drugs, thus reinforcing a protective and resilient TME. Abbreviations: (p)EMT-(partial) epithelial-to-mesenchymal transition; CSC-cancer stem cell; HGSOC-high-grade serous ovarian cancer; SASP-senescence-associated secretory phenotype; EV-extracellular vesicles (generated with BioRender, Toronto, Canada).

Extrinsic Tme Stressors Promote Intra-and Intercellular Adaptations
For example, in vivo, mechanical stress is felt and exerted by a growing tumor. External compressive stress is experienced by the tumor as the extracellular matrix (ECM), blood and lymphatic vessels resist tumor growth. Internal compressive stress is also developed as cancer cells proliferate within a confined space. Tensile stress is felt more strongly by cells along the periphery of the growing mass. Shear stress is experienced by cells exposed to the flow of interstitial fluid and, in the case of 40% of HGSOC cases, ascites fluid [8][9][10][11]. While our knowledge of the effects of mechanical stress on HGSOC is incomplete, it is established that mechanosensitive cancer cells, that is, cells that can internalize and respond to mechanical stimuli, can thrive or suffer under specific mechanical environments [7,10]. We have shown that HGSOC cells exhibit enhanced proliferation and chemoresistance on softer substrates [7]. Conversely, both 2D and 3D in vitro studies have demonstrated that stiffer substrates and high compressive forces can stunt HGSOC proliferation and promote apoptosis [6,7].
HGSOC cells alter both intracellular mechanisms and the surrounding stromal populations in response to mechanical stress. Indeed, HGSOC cells that are exposed to shear and compression stress in vitro develop stem cell-like properties with increased metastatic potential and chemoresistance [10]. HGSOC cells also have the ability to activate fibroblasts into cancer-associated fibroblasts (CAFs) either through enhanced paracrine signaling or direct physical interactions [12,13]. CAFs then play a crucial role in maintaining the TME by increasing the secretion of ECM components such as collagen and fibronectin or ECMremodeling proteins such as crosslinking proteins (lysyl oxidases) or matrix-degrading factors (matrix metalloproteinases (MMPs). CAFs, therefore, have the ability to change both the chemical and mechanical composition of the TME to enhance tumor growth and cancer cell invasion [14][15][16].
Similarly to mechanical stress, stress from nutrient depletion in the TME also has the ability to induce potent changes to cancer and stromal populations that have a lasting impact on disease progression. In the presence of extrinsic stressors such as the depletion of nutrients including glucose and amino acids, DNA damage, hypoxia, and reactive oxygen species (ROS), cellular homeostasis is compromised and thus triggers a series of responses that can ultimately drive cells to reorganize their bioenergetic process in a process called metabolic reprogramming. Examples of metabolic reprogramming include but are not limited to increased mitochondrial production, induction of the unfolded protein response (UPR), integrated stress response, and heat shock response [17,18].
The kind and degree of metabolic reprogramming which a cancer cell undergoes is tuned to the present stress. Heterogeneity in metabolic reprogramming within a single tumor is, therefore, highly likely given the range of local environments developed as a result of growth in 3D space. As cancer cells proliferate, recruit stromal cells, and promote matrix remodeling and angiogenesis, diffusion limitations are generated and gradients of nutrients and oxygen availability evolve as the volume and density of the tumor changes over time [6,19]. The metabolic landscape within a single tumor is, therefore, complex. While our understanding of metabolic reprogramming in ovarian cancer is incomplete, metabolic signatures that span cancer types have been identified and point to canonical pathways that are often aberrantly regulated.

Metabolic Reprogramming as a Priming Mechanism in Response to TME-Associated Stress
One such pathway is glycolysis and specifically the uptake of glucose, increase in glycolysis, and preferential fermentation to lactate despite the presence of oxygen, which is also known as the Warburg effect [20]. An increase in anaerobic glycolysis is critical under hypoxic conditions to maintain necessary ATP levels [21]. Alternatively, certain tumor subtypes exhibit a preference for oxidative phosphorylation (OXPHOS) [22]. In a study characterizing the metabolism of 127 clinical HGSOC samples and 14 ovarian cancer cell lines, Gentric et al. demonstrated that there was inherent metabolic heterogeneity among both samples and that they could be clustered into a high-OXPHOS and low-OXPHOS groups. High-OXPHOS samples relied on the tricarboxylic acid (TCA) cycle while low-OXPHOS cells mainly relied on glycolysis. The samples and cell lines categorized as high-OXPHOS had increased electron transport chain (ETC) synthesis, ATP production, basal respiration rate, and mitochondrial content. Importantly, these populations, labeled as high-OXPHOS, also exhibited enhanced chemosensitivity to taxanes and platinum-based drugs [22].
Cancer cells under nutrient deprivation and other environmental stressors are also known to have a particular reliance on glutamine and glutaminolysis. Indeed, glutamine not only serves as an alternative carbon source to fuel the TCA cycle and OXPHOS, but is also a key determinant of the ability of a cell to maintain redox homeostasis via the production of glutathione [23][24][25][26]. Critically, cancer cells also rely on glutamine for its role inducing and coordinating metabolic reprogramming across bioenergetic processes under the stress of nutrient deprivation or chemotherapy [27,28].
Upregulation of fatty acid (FA) uptake and metabolism have emerged as critical elements to metabolic reprogramming in HGSOC. Both the primary tumor site and metastatic niche of the omentum are abundant in FAs as a result of malignant peritoneal fluid buildup (ascites) and the secretome of adipocytes [29]. FAs can serve as alternative carbon sources to fuel the TCA cycle and OXPHOS for ATP when other carbon sources are not available [30][31][32][33]. FAs also serve as key secondary signaling molecules to enhance the proliferation and survival of cancer cells, a well-studied example being phosphatidylinositols and specifically PIP 3 and its role in the activation of the AKT-PI3K pathway [34]. Importantly, inhibition of FA uptake and metabolism greatly reduce HGSOC resistance to anoikis and overall progression [35,36].
Aberrant regulation of, and reliance on multiple bioenergetic and biosynthetic pathways provide cancer cells with alternative carbon sources to generate ATP and other critical molecules. Coordination of the induction and maintenance of these pathways is complex and essential to achieving cellular homeostasis. Autophagy is responsible for the degradation of compromised or excess proteins and organelles [37]. Briefly, autophagy proteins (ATGs) come together to form a double-membraned vesicle (autophagosome) labeled with the membrane-associated protein, LC3-II. LC3-II is then recognized by adaptor proteins which traffic specific substrates for degradation. Lysosomes then fuse with autophagosomes, a process regulated by SNARE proteins and small GTPases, and the engulfed substrates are broken down by lysosomal enzymes [38]. In the context of cancer under environmental stress, autophagy can generate pools of metabolites (for example, amino acids from proteins, FAs from lipids, and sugars from DNA) that feed into biosynthesis pathways (TCA cycle and glycolysis). By modulating available metabolite pools, autophagy regulates the impact any one pathway has on metabolic stability, thus conferring metabolic plasticity and overall resilience [37].
The selective autophagy of mitochondria, or mitophagy, provides another means of metabolic reprogramming. Mitochondria are tagged for degradation mainly when OXPHOS is compromised, which can be a consequence of the accumulation of ROS [39]. Oxidative stress is accrued when ROS levels surpass the capacity of antioxidant systems to remove ROS-a state often associated with hypoxia [40,41]. Mitophagy serves as such a mechanism to attenuate oxidative stress and reestablish redox homeostasis by reducing the production of ROS and the extent of oxidative damage [40,42]. In addition to mitophagy, mitochondrial fission and fusion can be modulated to reorganize and adapt mitochondrial networks in response to hypoxia. Indeed, HGSOC cells exposed to peritoneal-like hypoxic conditions had elevated levels of ROS and exhibited enhanced fragmentation [41]. Importantly, studies have shown that cancer cells primed with such mitochondrial stress have enhanced chemosensitivity [43,44]. Priming by oxidative stress has also been demonstrated to increase the apoptotic threshold by overexpressing prosurvival factors and thus conferring chemoresistance [45,46].
Environmental stressors promote metabolic reprogramming as an intracellular adaptation to cope with such stress. Extrinsic stress also enhances paracrine signaling which ultimately impacts the malignancy and resiliency of the TME [47]. The HGSOC secretome is an abundant source of cytokines, growth factors, and extracellular vesicles that guide critical proteins, transcription factors, and miRNAs through complex extracellular environments [47,48]. Buildup of such a malignant secretome is, unfortunately, common in late-stage HGSOC and referred to as ascites [47]. Although these proteins, nucleic acids, lipids, and vesicles are constantly being secreted, their release can be triggered and altered by multiple stimuli [11]. These can include TME stressors, such as hypoxia, interstitial pressure, and therapy-induced damage [49,50]. These stimuli alter intracellular pathways in ways that are reflected in the molecular content of the cytosol and biomaterials (proteins, RNAs, and miRNAs) that are packaged into multivesicular bodies and their secreted exosomes [51,52]. Therefore, differentially regulated miRNAs can serve as biomarkers for aggressive and even drug-resistant HGSOCs prior to the administration of chemotherapy.

Chemorefractory HGSOC Highlights the Priming Capabilities of the TME
The recommended chemotherapy treatment for advanced-stage HGSOC after diagnosis is a course of frontline drugs carboplatin and paclitaxel [53]. Platinum-based drugs form DNA adducts through crosslinking which prevents DNA synthesis and leads to the accumulation of double-strand breaks (DSBs). The inability to synthesize DNA or repair the resulting DNA damage can cause apoptosis [54]. Antimitotic agent paclitaxel functions by binding to β-tubulin subunits, thus stabilizing microtubules (MTs). Unable to depolymerize or reorganize MTs, mitosis is stalled as cells attempt to undergo cytokinesis, and apoptosis is subsequently activated [55]. Several modes of resistance have been identified in in vitro studies to either paclitaxel or platinum-based drugs, and studies using clinical samples have been critical in identifying the common traits of cancer cells; however, treatment efficacy and future drug development would greatly benefit from the ability to differentiate between resistant populations that are primed by the TME and those that are not. Critically, by characterizing 22 matched pre-and post-neoadjuvant chemotherapy-treated HGSOC patient samples, Zhang et al. demonstrated that treatment enriches subpopulations with an initially increased transcriptomic stress response and primes HGSOC cells to resist chemotherapy [56]. Kan et al. similarly identified that relapseinitiating HGSOC cells can largely originate from a subpopulation early in tumorigenesis with a high-stress signature. Relapse-initiating cells also developed over the course of HGSOC progression [57]. By identifying the intrinsically resistant populations in refractory HGSOC, subsequent chemotherapeutic interventions can be designed and optimized to target known vulnerabilities of these distinct populations.
In this review, we discuss how the prominent TME stressor of hypoxia and therapyinduced stress drive HGSOC cell populations to secrete a unique secretome content that both fosters chemoresistant populations and promotes invasive phenotypes in neighboring populations.

Hypoxia Confers Resistance in Refractory HGSOC
While 25% of HGSOC cases are refractory and exhibit intrinsic resistance to chemotherapies, studies with a specific focus on refractory HGSOC are limited, with most using treatment-naïve samples [58]. By characterizing pre-and post-chemotherapy HGSOC patient samples using single-cell RNAseq, whole genome sequencing, and DNA copy number analysis, groups have found either no recurrent genomic changes in subpopulations primed to endure chemotherapy, BRCA1/2 mutations or amplification of 19q12, containing cyclin E (CCNE1) ( Table 2) [56,58,59].
Indeed, these multipronged studies emphasize that intrinsically resistant subpopulations are most unique in their transcriptional profiles. Furthermore, such profiles consistently describe a stress-associated state which primes cancer cells to endure exposure to chemotherapy. These results highlight the potent effects of environmental stressors and the role of subsequent coping mechanisms in generating a resilient HGSOC population.
One of the most potent stressors in the TME is hypoxia. As cancer cells proliferate and recruit neighboring cells, the TME evolves in 3D space and spatial heterogeneity is developed. As a result, oxygen gradients are formed by diffusion limitations throughout the 3D growth [6,19], thereby creating a range of local environments. It follows, then, that the responses cells employ to cope with stress are nonuniform and produce heterogeneity in proliferative capability, migratory probability, metabolism, and chemoresistance.
In normoxic conditions, the hypoxia-inducible factor alpha (HIF-α) subunit is hydroxylated and recognized by the von Hippel-Lindau tumor suppressor protein (pVHL) for degradation via the ubiquitin-proteasome pathway. With lower concentrations of oxygen, the degradation of HIF-α is stunted; it then dimerizes with the constitutive HIF-1β. The HIF complex can then act upon a wide range of target genes [60,61]. While we continue reviewing how chemoresistance is developed, in part, as a result of HIF-1α activity, we acknowledge that HIF-1α activity is not exclusively triggered by low oxygen concentrations. Several studies have revealed oxygen-independent pathways of HIF-1α activation such as the accumulation of metabolites including lactate, pyruvate, and succinate [62][63][64][65][66].
Clinically, overexpression of the HIF-1α isomer regardless of the p53 status highly correlates with poor prognosis and resistance to platinum-based drugs in ovarian cancer [67]. Furthermore, exposure to hypoxia either prior to or in conjunction with treatment increases resistance to both frontline chemotherapeutics cisplatin and paclitaxel [68,69]. While several mechanisms are employed by cancer cells to cope with hypoxic conditions, a consistent overarching response is to adopt a flexible stem-like phenotype-a process largely regulated by the HIF pathway [70,71]. The epithelial-to-mesenchymal transition (EMT) broadly describes a series of molecular events whereby differentiated epithelial cells elongate and lose their cellular adhesions and develop a less differentiated and more motile mesenchymal phenotype with increased extracellular matrix adhesions [71]. As such, EMT is implicated in stem cell interactions, embryogenesis, wound healing, and cancer metastasis [72,73]. In the context of ovarian cancer, EMT is critical for the ability of cancer cells to disassociate from the primary site, resist anoikis, and migrate to the secondary site. Once cancer cells reach the secondary tumor site, they undergo a mesenchymal-to-epithelial transition (MET) to establish cellular adhesions important in the growth of metastatic tumors [74,75].
Given the range of oxygen availability throughout the TME, cancer cells that are exposed to lower oxygen concentrations are driven towards EMT. However, depending on the tissue and the context of extracellular signals, some epithelial cancer cells lose only some epithelial characteristics, demonstrating both mesenchymal and epithelial characteristics [76]. This phenomenon is denoted as partial EMT (pEMT) and is frequently identified across many cancer types and depends on the frequency and degree of hypoxia exposure [68,77,78].
HIF-regulated (p)EMT serves as a mechanism to induce and sustain a flexible resilient cell state. Indeed, hypoxia has been shown to prime cancer cells with a ROS-resistant phenotype which is sustained after the initial exposure and contributes to survival during metastasis [79]. Furthermore, HIF-1α induces autophagy and is critical for the maintenance of cancer stem cell (CSC) populations in ovarian cancer [80,81]. HIF-1α can also directly promote CSC traits by activating the nuclear factor (NF)-κB transcription factor, which is critical in inflammation [82]. In addition to HIF-1α, other signaling axes such as NOTCH1-SOX2 and the UPR have also been identified as the key modulators of the induction of autophagy and CSC maintenance under hypoxic conditions [83,84]. One of the advantages of adopting a stem cell-like phenotype is developing plastic metabolism through the induction of autophagy and cancer stem cell-like phenotypes. While the precise cascade of alterations to bioenergetic pathways in ovarian CSCs is still being unraveled, several studies have demonstrated that ovarian CSCs have increased glycolysis, OXPHOS, and altered lipid metabolism which can then, in turn, help them resist nutrient deprivation to survive adverse conditions in the tumor microenvironment [85][86][87]. Lastly, ovarian CSCs that are primed by surviving hypoxic conditions are more aggressive when returned to normoxic conditions as evidenced by enhanced proliferation, migration, and colony formation [88].
Studies have demonstrated that hypoxic conditions can also promote proliferation and inhibit apoptosis in ovarian cancer [89]. STAT3 is implicated in several proliferation and apoptosis signaling axes and is often aberrantly activated in ovarian cancer [90]. STAT3 activity is induced by hypoxia and not only regulates proliferation under hypoxic conditions, but also confers both cisplatin and paclitaxel resistance, in part by priming ovarian cancer stem cells to survive in adverse conditions [49,91,92]. Furthermore, silencing STAT3 via siRNA or a chemical inhibitor reverses hypoxia-dependent resistance [93,94].
Overall, hypoxia elicits a metabolically flexible stem cell-like state by the induction of EMT. The subsequent induction of autophagy and metabolic reprogramming are not only critical to the maintenance of such a cell state, but also confer resistance to the present environmental stress and future stress of chemotherapy. Herein, we discuss the associated changes in the secretome of HGSOC cells primed by the stress of their TME.

Hypoxia Alters HGSOC Secretome Profile
The tumor secretome consists of a wide range of proteins, growth factors, and metabolites that play important roles in cell-cell and cell-matrix interactions [95]. Aberrant autocrine and paracrine factors found in the tumor secretome can serve as prognostic and diagnostic HGSOC markers [96]. Hallmark TME stresses (i.e., highly dysfunctional vasculature with leaky, compressed blood vessels and poor microcirculation, which results in reduced oxygen concentrations) can result in heterogenous secretome profiles [97,98]. Secretome changes are of particular importance in the context of HGSOC because patients present with large accumulations of ascites fluid (potent source of cell-secreted factors or the secretome) that are unique to these cancers and metabolic conditions in the peritoneal cavity.
Clinical studies have found that ascites components that are largely altered in patients with chemosensitive tumors vs. patients with drug-resistant tumors include (i) cytokines, (ii) growth factors, and (iii) extracellular vesicles (EVs) [99][100][101][102][103]. EVs-i.e., exosomes, microvesicles, and oncosomes-are highly involved and play a critical role in transferring drug-resistance phenotypes during cell-cell communication. Ascites fluids harvested from HGSOC patients are rich with circulating vesicles and show atypical levels of secreted cytokines and growth factors that are linked to protumorigenic pathways. Numerous cytokines and growth factors, such as interleukin 6 (IL-6), interleukin 8 (IL-8), transforming growth factor-beta (TGF-β), epidermal growth factor (EGF), and vascular endothelial growth factor (VEGF), are upregulated and can alter the TME to promote chemoresistance by activating antiapoptotic and prosurvival signaling pathways [104][105][106]. Clinical studies have revealed that patient ascites contain~40-500 times more proinflammatory cytokines compared to serum [107]. EVs have more recently emerged as a novel mechanism of enhancing drug resistance via cell-cell communication [108]. These circulating vesicles encapsulate and transfer cellular cargo, along with cytokines, growth factors, and other non-protein content, that can alter recipient cells to develop potent drug-resistant phenotypes [109].

Cytokines
Cytokines-i.e., IL-6, IL-8, IL-11, IL-27, IL-31-are small immunological proteins important in autocrine, paracrine, and endocrine signaling processes that influence inflammation, cell growth and proliferation, cell and matrix interactions, and disease progression in cancer [110]. IL-11, IL-27, and IL-31 are members of the IL-6 family. IL-11 has been reported as an important anti-inflammatory or tumor-promoting cytokine. IL-11 has also been particularly linked to poor prognosis in cancers that possess epithelial traits [99]. However, the specific role of IL-11 in HGSOC remains unclear as IL-11 expression is low in this specific cancer [111]. Similarly to IL-11, IL-27 takes on a dual role in the context of the TME. However, it has been highlighted that IL-27 suppresses SKOV3 cell proliferation by simultaneously activating STAT3 and inhibiting the Akt pathway [112]. Elevated IL-31 levels have been correlated to poor prognosis [113]. Further, IL-31 has been shown to enhance mesenchymal HGSOC cell phenotypes such as proliferation, migration, invasion, and survival. IL-17 is a proinflammatory cytokine that is largely produced by T helper cells and macrophages. Studies have shown that IL-17 assists the renewal of cancer stem-like cells, thereby driving HGSOC tumorigenesis [114].
Cytokines are secreted by immune cells and other cells in the tumor, including HGSOC cells and surrounding primary TME cells (i.e., stromal, endothelial, epithelial, mesothelial) [11,100]. Cytokines mediate intercellular communication between immune and nonimmune cells in the TME. This communication can lead to T cell activation and macrophage differentiation [115]. It has been further established that individual inflammatory cytokines or their cohorts work collectively in activating potent cell phenotypes that assist in conferring chemotherapy resistance and promoting ovarian cancer immune evasion. Tumor cells release these immunosuppressive cytokines, which can activate tumor-associated macrophages and reprogram cells that used to be immunostimulatory to immunosuppressive [116]. These cells then, in turn, secrete immunoinhibitory cytokines that support tumor cell survival, metastasis, and cytotoxic T cell recruitment [117]. In fact, high-grade serous HGSOC are often regarded as immunologically "cold" tumors.
These cytokines serve as important bio-or predictive markers. Numerous in vitro studies examining cell types, such as IGROV-1, PEO1, SKOV3, and OVCAR3, have reported that tumor necrosis factor-alpha (TNF-α), IL-6, and IL-8 act as potent paracrine and autocrine signaling molecules that regulate cancer cell invasion, proliferation, and bulk tumor growth. These phenotypic differences contribute directly to promoting intrinsic chemoresistance [118,119].
Tumor hypoxia can activate transcription factors that are responsible for elevating intracellular cytokine expression and release profiles. Elevated cytokine release from HGSOC TME cells amplifies the autocrine and paracrine feedback loop, which is involved in immune cell recruitment. Cytokines are potent mediators of immune cell homing to tumors and metastatic tissues. This subsequently highlights that hypoxia-induced cytokines serve as proinflammatory factors that are critical in establishing chemorefractory phenotypes. For example, in vitro studies have shown that hypoxia directly modulates activator protein-1 (AP-1) and NF-kβ in HGSOC cells. AP-1 and NF-kβ conjunctively play important roles in secreting higher IL-8 levels in SKOV and Hey8 cell lines [120]. Hypoxic conditions, low pH, and poor vascularization in the TME further contribute to the abnormal cytokine profiles. HGSOC cells cultured in in vitro acidic conditions (pH 6.6) demonstrated increased IL-8 secretion, which was associated with the activation of AP-1 and NF-kβ and the development of chemoresistance [121].
TNF-α also plays a key role in regulating local and distal invasion, angiogenesis, and metastasis in numerous cancers, particularly in HGSOC. Similarly to IL-6 and IL-8 profiles, TNF-α is found at high circulating concentrations under hypoxic conditions and in HGSOC patient TMEs. TNF-α plays pivotal roles in autocrine and paracrine signaling mechanisms that activate pathways important in invasive and chemoresistant cancer cell behaviors. For example, TNF-α activates NF-kβ and further canonical pathways [122]. Activation of NF-kβ subsequently reduces the p53 transcription factor activity; p53 is a tumor suppressor that halts cell cycle progression and cell division in response to DNA damage or genetic instability [123]. Mutations in p53 and loss of functional p53 activity trigger invasive and chemoresistant cell behavior [124].
Proinflammatory cytokines secreted in response to hypoxic conditions are important paracrine and autocrine factors that regulate signaling pathways responsible for cancer cell proliferation, intrinsic chemoresistance, and invasion in local and distal tumor sites.

Growth Factors
The HGSOC secretome is enriched with growth factors that support and maintain cells that have detached from the primary tumor and attached at the secondary site. The repertoire of growth factors and growth factor receptors is altered between HGSOC patient and normal donor tissues and blood and can aid in conferring chemoresistance [124,125]. Anticancer drugs and therapies not only influence the growth factor profiles in the secretome, but potent hypoxic conditions also contribute to this abnormal profile of circulating growth factors [126]. As a homeostatic response to hypoxic TME conditions, HGSOC cells often secrete increased concentrations of EGF. EGF further maintains tumor hypoxia by elevating HIF-1α expression [127]. Simultaneous in vitro elevation of EGF and HIF-1α promote EMT phenotypes in SKOV3 and OVCAR5 cells, which are associated with reduced E-cadherin and increased Snail and Slug expression [127]. Further, HIF-1α increases VEGF expression [128]. VEGF is essential in angiogenesis and ultimately promotes tumorigenesis through cell migration, proliferation, and survival [129]. Overall, hypoxic conditions activate transcription factors that trigger aberrant growth factor secretion.

EVs
TME stresses, particularly hypoxia, increase EV biogenesis from cancer cells, while also regulating EV protein and miRNA synthesis; yet, it is not well-understood how hypoxia directly accelerates EV secretion. A substantial body of in vitro evidence has linked HIF-1α activation to elevated proteins responsible for EV secretion, such as Rab proteins that regulate intracellular vesicle transport [130,131].
A more recent in vitro study has further demonstrated that hypoxia triggers CAOV3 cells to release small EVs that confer carboplatin resistance to neighboring cancer cells. Specifically, EVs released under these hypoxic conditions alter glycolytic and fatty acid synthesis pathways that support chemoresistance in recipient, normoxic HGSOC cells [103]. More importantly, a cohort of these identified EV-associated glycolytic proteins were patient-specific and can be further used as predictive markers for refractory HGSOC [103].

Hypoxia Alters HGSOC Exosomes
Exosomes are a subcategory of EVs (40-160 nm diameter) that play crucial roles in intercellular communication and chemoresistance. Exosomes play pivotal roles in cell-cell interactions, often altering physical phenotypes and signaling pathways in recipient cells. Their cholesterol-rich membranes allow exosomes to serve as stable vehicles during intercellular communication and prevent enzymatic degradation of sensitive protein and RNA content. These exosomes are packaged with functional biomolecules, such as proteins, RNAs, and miRNAs, during intraluminal vesicle transport; the exosome content is highly representative of parent cells (i.e., drug-resistant HGSOC cells) and HGSOC tumor microenvironment [132]. This allows exosomes and encapsulated cargo to act as useful biomarkers for malignant and invasive HGSOCs [132]. In vitro evidence with human cervix carcinoma cells has highlighted that chemoresistance alters proteins that are essential in the endosomal pathway, subsequently regulating exosome biogenesis [133].
Additionally, hallmark primary TME conditions that accompany chemorefractory development, such as hypoxia and low pH, alter and accelerate exosome release mechanisms. In vitro cancer cells (i.e., various metastatic grades of HGSOC, breast, pancreatic, and lung cancer cells) and cancer-associated stromal cells (i.e., fibroblasts, stem cells, myeloid cells) cultured under hypoxic conditions secreted more exosomes than cells cultured under normoxic conditions. This rapid exosome secretion may be attributed to the upregulation or activation of transcription factor genes under hypoxic stress [49,[134][135][136]. These transcription factors, including tetraspanins, soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), tethering proteins, and Rabs, regulated exosome biogenesis and secretion proteins. HIF-1α activates Rab22a, an essential protein during exosome secretion. Rab22a colocalizes to budding vesicular membranes [137]. Actins, along with other major cytoskeletal proteins, are critical in transporting extracellular vesi-cles from the cytoplasm to the plasma membrane. Hypoxia can also alter actin filament organization. In vitro studies with A2780 cells have shown that hypoxia positively induces ROCK, a regulator of actin dynamics, to increase exosome biogenesis and secretion [138]. These secreted exosomes encapsulated aberrant levels of HIF-1α, interleukins, and matrix degradation enzymes. Exosomes subsequently propagated EMT phenotypes and conferred chemorefractory resistance in recipient cells [139].

Acquired Chemoresistance Is Achieved through Drug-Specific Adaptations and Induction of Therapy-Induced Senescence
Cancer cells that manage to cope with hostile TME conditions are more likely to survive future chemotherapeutic treatments. The TME can increase the apoptotic threshold in cancer cells as well as induce a dormant stem-like phenotype. Additionally, the TME promotes modifications in the HGSOC secretome which fosters a protective phenotype in neighboring cells and reinforces resistance. Evasion of subsequent chemotherapy treatment can promote additional drug-specific modes of resistance.
Ovarian cancer cells that survive paclitaxel treatment often develop altered cytoskeletal dynamics [55]. Cells that survive paclitaxel have been shown to have molecular alterations in tubulin variants and post-translational tubulin modifications that hinder paclitaxel from properly binding MTs [140]. Dynamics of MT growth and catastrophe are also altered in response to paclitaxel treatment and have been shown to enable ovarian cancer cells to more quickly attach and migrate on polyacrylamide substrates through increased traction stress generation [7]. In addition to cytoskeletal changes, HGSOC cells can employ efflux pumps to cope with paclitaxel treatment. Paclitaxel is a substrate of efflux pump P-glycoprotein (Pgp) [141,142]. P-gp is encoded by gene MDR1, and across several studies, MDR1 expression or P-gp structure has been enhanced or modified, respectively, in ovarian cancer cells after chemotherapy [1,59].

Therapy-Induced Senescence and Escape as a Mechanism for Recurrent Disease
Paclitaxel and platinum-based drugs can also elicit similar changes seen in response to TME stressors [140,[143][144][145][146][147]. Post-chemotherapeutic intervention, these collective changes, including the induction of autophagy, metabolic reprogramming, EMT, and enhanced paracrine signaling, are indicators of therapy-induced senescence (TIS). In addition to the aforementioned changes, cancer cells with a TIS profile have characteristics associated with cellular senescence such as cell cycle arrest and compromised nuclear integrity. TIS has emerged as a key contributor to dormant tumors and recurrent disease [148]. We should note that while dormancy has been associated with a quiescent cell profile, senescence more accurately describes the overall trajectory and molecular changes sustained by cells that escape dormancy and fuel recurrent disease. A quiescent cell will revert to a prestress phenotype and resume proliferation once the stressor (intrinsic or extrinsic) has been removed; however, a senescent cell does not necessarily revert or begin to proliferate under favorable conditions and, importantly, retains some of the adaptations developed in response to the stress [148,149].
Cancer cells can also undergo senescence in the absence of therapy, in a process named oncogene-induced senescence (OIS). OIS is driven by oncogenic stress (for example, DNA damage) that is generated by the aberrant expression or regulation of oncogenes or tumor suppressors (including, but not limited to, Ras, Raf, Akt, PTEN) [150]. The prerequisite to both OIS and TIS is activation of the DNA damage repair (DDR) response [151]. While in OIS the DDR response is activated to cope with DNA damage caused by telomere attrition and loss of tumor suppressors, the DNA damage leading to TIS can be caused directly by DNA crosslinking properties of platinum-based drugs (cisplatin or carboplatin) as well as the associated DNA damage caused by prolonged paclitaxel-induced mitotic arrest [151][152][153][154].
Defective DDR is a hallmark of late-stage ovarian cancers. Platinum-based drugs elicit DNA damage and require cancer cells to either enhance or modify the existing DNA damage repair (DDR) mechanisms [155]. For example, ovarian cancer cells that survive carboplatin treatment often have defective nonhomologous end joining (NHEJ) repair mechanisms [156]. Tumor suppressor genes BRAC1/2 which are critical in the DDR also have consistent mutations in therapy-resistant patients [59]. Upstream of DDR mechanisms, epigenetic modifications such as DNA methylation and platination are also implicated in cisplatin resistance [155,157].
Cells that are exposed to high concentrations of anticancer drugs may undergo mitotic slippage or endoreplication to form polyploid giant cancer cells (PGCCs), which are multinucleated cancer cells that have high DNA content and multidrug resistance [153,158]. The PGCC phenotype is associated TIS; thus, PGCCs remain metabolically active but no longer undergo mitosis. PGCCs are capable of undergoing amitotic division through depolyploidization, which leads to the formation of diploid cancer cells that are capable of metastasis. These PGCC daughter cells also acquire the PGCC drug-resistant phenotype [153].
Whether mechanisms promoted by the TME, such as EMT, and TIS are mutually exclusive or molecularly linked is unclear; however, recent evidence demonstrates that they have several shared regulators and confer chemoresistance to cancer cells [159]. The implications of TIS and senescence more broadly in cancer progression are complex as there is evidence that suggests both pro-and antitumorigenic effects [151]. While senescence can stunt the proliferation of cancer cells, such dormancy can be protective by decreasing the efficacy of antimitotic drugs. Conversely, escape from dormancy, a process that relies on EMT, greatly contributes to disease recurrence [160]. TIS-associated induction of autophagy and stemness have been shown to drive disease recurrence [148,161]. Importantly, TIS promotes potent changes in the secretome of cancer and stromal populations known as the senescence-associated secretory phenotype (SASP) [162]. Herein, we discuss how the HGSOC SASP bolsters chemoresistance in cancer cells and fosters a protective TME.
2.6. Therapy-Induced Chemoresistance Alters the HGSOC Secretome HGSOC cells exposed to chemotherapeutics release unique secretome components that can confer chemoresistance to surrounding cells and can assist in developing a more invasive TME. Cells that survive therapeutic taxane or platinum-based drugs through TIS can further activate cells to secrete a more potent senescence-associated secretome that aids in chemoresistance development [50]. Similarly to other senescent cells, tumor cells that exhibit TIS remain metabolically active and develop altered secretome profiles. This unique secretome promotes chemoresistance to neighboring tumor-associated cells through the production of cytokines, growth factors, and EVs that promote cell survival.

Cytokines
Women diagnosed with HGSOC have consistently shown elevated circulating levels of IL-6, IL-8, and TNF-α. Clinical data have indicated that IL-6 and IL-8 are significantly amplified between benign ovarian tumors and HGSOCs [163,164]. Studies have shown that innate and activated immune cells are largely responsible for cytokine secretion. Immune cells that contribute to cytokine production include macrophages, T cells (i.e., CD8+ and CD4+), lymphocytes, and natural killer cells [165]. IL-6 and IL-8 are also upregulated in many senescent cells and play critical roles in supporting inflammatory environments, such as the primary HGSOC TME. Additionally, HGSOC cells that are cocultured with other cells in the HGSOC TME have been reported to secrete aberrant levels of IL-6 and IL-8. Several of these cells include SKOV3, OVCAR3, OVCAR4, IGROV-1, and HEYA8 [99,[166][167][168]. Similarly, senescent IMR90, WI-38, and MSCs and cancer-associated fibroblasts also contribute to amplified cytokine secretion in the primary TME [169][170][171]. IL-6 and IL-8 are highly recognized as critical SASP components and are involved in many pathways that modulate TME stresses and chemoresistance; thus, our review focuses on IL-6 and IL-8.
In vitro studies of IL-6 and IL-8 in HGSOC cells, such as OVCAR3, CaOV3, and SKOV3, have been related to malignant cancer cell behaviors. Induced IL-6 expression via recombination and transfection approaches in non-IL-6-expressing A2780 cells propagated these cells to develop drug-resistant phenotypes [104]. This chemoresistance subsequently enhanced drug resistance genes, such as multidrug resistance 1 (MDR1) and Glutathione S-transferase pi (GSTpi), apoptosis inhibitory proteins, and proteins associated with cell proliferation pathways [104]. Conversely, knockdown of endogenous IL-6 expression in IL-6-overexpressing SKOV3 cells amplified anticancer drug sensitivity [104].
IL-6 can also advance chemoresistance via STAT3 activation. STAT3 is required for HG-SOC cell migration and has been established in vitro (i.e., SKOV3 and A2780) to promote expression of proteins that are associated with mesenchymal phenotypes. It also upregulates survival proteins, such as B cell lymphoma-2 (BCL-2) and survivin, that advance chemotherapy resistance [172].

Growth Factors
Many growth factors are overexpressed in HGSOC and play critical roles in ovarian cancer progression and the development of chemoresistant phenotypes. Along with cytokines, many of these growth factors are upregulated in the SASP and exacerbate the alterations in aggressive and chemoresistant HGSOC cell phenotypes. For example, overexpression of the epidermal growth factor receptor (EGFR) and its respective ligands are linked to therapy-resistant HGSOC cell populations [173]. Upregulation of EGFR mediates pathways and activates transcription factors that influence invasive cell behavior, such as survival, proliferation, and migration. Cisplatin-resistant HGSOC cells promote EGFR activity. EGFR signaling through STAT3 led to increased in vitro A2780 cell proliferation, viability, colony formation, and invasive migration [174]. Aberrant EGFR and STAT3 in cisplatin-resistant cells altered cytoskeletal expression and intercellular localization. These cells exhibited increased levels of cortactin and F-actin, where F-actin localized predominately to protrusions [174]. This in vitro work further reported that inhibition of hyperactive EGFR led to cisplatin sensitivity by reducing VEGF and survivin activities. Therefore, genes activated via EGFR signaling (i.e., STAT3) could serve as effective targets for therapeutic strategies in combating chemoresistant cancers [174].
Dysregulation of TGF-β1 and TGF-β3 and subsequent downstream canonical and noncanonical pathways have been shown to be activated in refractory ovarian cancer [175]. In fact, TGF-β isoforms are often amplified in the secretome of women who present with therapy-resistant HGSOC cases. Extracellular stress and anticancer therapies modulate TGFβ secretion. It is well-known that TME stress can induce TGF-β-mediated EMT response in HGSOC cells [176]. In vitro studies have also highlighted that inhibiting TGF-β activity via its receptors leads to cisplatin sensitivity in SKOV3 cells [177]. Conversely, cisplatinresistant HGSOC cells had elevated TGFβR2 levels compared to sensitive cancer cells; this underscores the importance of the TGF-β pathway in chemoresistant HGSOC [177]. TGF-β ligands modulate numerous processes, such as cell growth, differentiation, and SMADactivated apoptosis. Dysfunctional TGF-β activity in chemoresistant HGSOC cells (A2780) can lead to the activation of downstream genes that are elevated in HGSOCs [178,179].

EVs
Anticancer therapy induces irregular intracellular protein trafficking and EV secretion. In vitro studies have shown that early lysosomes of HGSOC cells pretreated with chemotherapeutic drugs are heavily concentrated with cisplatin [180]. These cancer cells readily export these drugs via endosomal vesicle secretion.
Upon acquiring chemoresistance, HGSOC cells maintain aberrant EV biogenesis and secretion. Transmission electron micrographs showed the cytoplasm of resistant HGSOC cells is more heavily concentrated with EVs than that of chemosensitive HGSOC cells [181]. Chemoresistant HGSOC cell-secreted EVs also encapsulate numerous cytokines and growth factors that were previously highlighted in this review to be important in cell-cell communication and aggressive and chemoresistant cell phenotypes.
Safaei et al. highlighted that in vitro chemoresistant HGSOC cells, OV-2008 cells, exhibited unique EV secretion patterns that sustained their chemoresistant phenotypes by increasing the expression of transporter proteins important in effluxing multiple drugs [180]. These transporter proteins were multidrug resistance-associated protein 2 (MRP2), ATP7A, and ATP7B. They further demonstrated that EVs from chemoresistant cells exhibited a 4.9-fold higher cisplatin content compared to EVs from chemosensitive cells [180], strongly suggesting that EVs play critical roles in maintaining chemoresistant phenotypes.
EVs released by chemoresistant IGROV-1 cells can induce invasion and drug resistance in neighboring bystander cells. HGSOC cells pretreated with EVs from chemoresistant HGSOC cells had increased viability after a subsequent drug treatment, implying that treatment resistance had been transferred with EV treatment [182]. Conversely, cells that were treated with EV uptake inhibitors, such as heparin, dynasore, and amiloride, prior to EV exchange remained chemosensitive. Several proteins that were responsible for chemotherapy adaptation and cell invasion included p38α, p53, and factors involved in JNK signaling processes; p38α and p53 were downregulated, which subsequently contributed to decreased drug sensitivity; c-Jun N-terminal kinase (JNK) pathway activation has been linked to elevated cell invasion and migration. This in vitro study indicated that these EVs could activate prosurvival pathways.

Role of Exosomes in Developing Chemoresistance
Circulating tumor exosomes serve as critical mediators of chemoresistance in the ovarian TME. When exosomes were exchanged in vitro between therapy-resistant OV-CAR10 and therapy-sensitive A2780 cells, the sensitive cells developed more invasive and therapy-resistant phenotypes, indicating that phenotypical characteristics could be transferred via exosome content. HGSOC cells pretreated with exosomes harvested from platinum-resistant HGSOC cell lines showed a twofold increase in cell viability after carboplatin treatment. These platinum-resistant ovarian cancer cells further exhibited SMAD4 mutations, which led to some content alterations. In vitro exosome exchange led to increased EMT phenotypes (i.e., increased mesenchymal markers, such as N-cadherin and Zeb1) and induced platinum-resistant phenotypes in once therapy-sensitive cells [183].
Chemoresistant HGSOC cell exosomes can also drive more potent invasive cellular behaviors by promoting angiogenesis. Li et al. showed that exosomes derived from chemoresistant SKOV3 cells increased human umbilical vein endothelial cell (HUVEC) proliferation, migration, and invasion [108]. More strikingly, these chemoresistant cellderived exosomes led HUVECs to form capillary-like tubes that support angiogenesis. They also reported that chemoresistant cell-released exosomes showed a significantly higher miRNA-130 expression compared to exosomes released by chemosensitive cells. Therefore, exosome miRNA-130 could promote angiogenic development in chemoresistant HGSOC cells [108].

Changes in miRNAs
MicroRNAs are small noncoding RNAs (19-25 nucleotides) that regulate post-transcriptional gene expression through complementary binding and degradation of mRNA and/or translational repression [184]. MicroRNAs are highly conserved across species; however, alterations in their nucleotide sequences or changes in target mRNA can prevent binding to alter gene regulation. Differences in miRNA expression can also alter gene regulation. Numerous in vitro, preclinical, and clinical studies have shown that differential miRNA expression is critical in metastatic initiation, progression, and dissemination [185]. The repertoire of miRNAs in HGSOC samples can be determined from tissue and fluid biopsy samples and can serve as a miRNA signature of various types of ovarian cancers (serous epithelial, mucinous, clear cell). This miRNA repertoire includes critical biomarkers for different cancer types, disease stage, and patient prognosis.
Early clinical studies focused on examining miRNA content in blood and surrounding intraperitoneal fluids for early HGSOC diagnosis. Blood cells and circulating soluble factors that were also present in these samples made it difficult to study the tumor cell miRNA content. Rapid enzymatic miRNA degradation made it further challenging to examine how differentially regulated levels correlated with the disease state. Thus, miRNA content in isolated exosomes has been used to overcome these limitations [186][187][188]. The cholesterolrich exosome membrane protects sensitive miRNAs from degradation. Circulating exosome miRNA content from the TME more accurately represents molecular phenotypes of tumorassociated cells compared to other signaling molecules.
Numerous factors can alter miRNA content in secreted exosomes, ranging from cellular heterogeneity to differences in the inherent TME stress factors that contribute to exosome biogenesis and secretion. For example, tumor hypoxia enriches miRNA-181d-5p in secreted SKOV3 exosomes [189], and these hypoxia-induced exosomes enhance tumor cell migration and invasion in vitro. Another example of how miRNA content is altered in late-stage tumor-derived exosomes is in the miRNA-200 cohort, a well-acknowledged HGSOC miRNA family, which is elevated in chemoresistant HGSOC patients [190,191]. Conversely, Let-7i/g expression is decreased in chemoresistant HGSOC patients. Reduced levels of Let-7i/g amplified EMT and activated prosurvival pathways in the cells treated with therapy-resistant exosomes. Table 1 further details important miRNAs along with specific gene targets that are significantly regulated that lead to chemorefractory and chemoresistant HGSOC behaviors. Thus, exosome miRNAs are critical HGSOC biomarkers. Table 1. Unique and overlapping miRNA profiles in refractory HGSOC. The table highlights the miR-NAs and the respective targets/function that were differentially regulated under hypoxic conditions or after chemotherapeutic HGSOC treatment; miRNAs in green represent those that are upregulated and miRNAs in red represent those that are downregulated.

Conclusions
Tumor recurrence is a significant hurdle to overcome across cancers, but is especially poignant in HGSOC. Therapy-induced chemoresistance is well-acknowledged and investigated; yet, the mechanisms describing how properties of the TME lead to cellular stress responses that contribute to chemoresistance in refractory disease remain unclear. One of the main properties of the TME that confers chemoresistance is hypoxia. Hypoxia drives HGSOC populations to develop (p)EMT and CSC profiles. Importantly, the establishment and maintenance of CSC and (p)EMT phenotypes are intimately tied to the induction of autophagy and downstream metabolic reprogramming. Collectively, these events result in metabolically flexible and resilient cancer cells.
It is these populations that have endured environmental stress (hypoxia being only one of many in the TME) that are best equipped to overcome subsequent exposure to stress such as chemotherapy. Zhang et al. identified several gene signatures among treatment-naïve HGSOC populations such as proliferative DNA repair, RNA processing, TCA cycle, among others. Strikingly, however, HGSOC populations post-neoadjuvant chemotherapy were enriched for a stress-associated profile (see Table 2) [56]. Pools of HGSOC cells with a similar stress profile have also been identified as likely sources of relapse-initiating cells [57]. These are two examples of robust studies which were able to differentiate between refractory and resistant HGSOC populations by following the genesis and trajectory of resistanceassociated profiles before and after treatment. These studies are necessary given the significant gap in our understanding of the unique characteristics distinguishing between chemorefractory and chemoresistant HGSOC despite the prevalence of both in the clinic. Future studies that are able to identify differentiating properties of chemorefractory and chemoresistant HGSOC can lead to the development of precise treatments for each disease that exploit their unique vulnerabilities. HIFs [60] Tetraspanins [137] SNAREs [137] Rabs [137] Translational/post-translational AP-1 [120] β-tubulin III [224] STAT3 [90] NF-kB [120] JNK [182] Akt/mTOR [113]  With regard to chemorefractory HGSOC, key regulators of metabolic reprogramming are potent candidates for future anticancer agents. As we have covered here, metabolic reprogramming is an essential process that governs a cancer cell's ability to cope with a wide range of environmental stressors, especially hypoxia. Additionally, metabolic reprogramming and the resulting resilient populations are critical contributors to recurrent disease.
There are several approved anticancer agents that target cancer metabolism (e.g., methotrexate, 5-fluorouracil, gemcitabine); however, there are metabolic inhibitors currently in clinical trials that target more newly appreciated key elements of metabolic reprogramming such as glutaminase (CB-839, IPN60090), lactate symporters (AZD-3965), and fatty acid synthesis (TVB-2640) [225]. These metabolic inhibitors are promising therapies, especially for refractory disease. Yet, in order to develop and accurately gauge the efficacy of these therapies against refractory HGSOC, we must drastically improve our diagnostic capabilities-otherwise we will continue to enrich resistant cancer populations with the treatment of standard chemotherapy.
A candidate feature of HGSOC that would aid in the detection and differentiation of chemorefractory and chemoresistant populations is the secretome. The secretome is greatly influenced by hypoxia since low oxygen concentrations assist in the development of other protumorigenic traits of the TME such as low pH and leaky vasculature. These factors can amplify the secretion of soluble factors and the expression of transcriptional regulators responsible for chemoresistance. Importantly, studies have highlighted that hypoxia modulates pathways necessary for HGSOC cell vesicle synthesis and secretion. Hypoxia-induced exosomes are potent paracrine factors that can induce invasive and chemoresistant phenotypes in neighboring stromal cells and cancer cells. Studies looking at how specific TME stresses contribute to chemoresistant and refractory ovarian cancers through alterations in their secretome profiles are critical in understanding the mechanisms contributing to chemoresistance. Many miRNAs are differentially expressed in chemoresistant phenotypes; however, only a few have been directly linked to chemoresistance [202]. Furthermore, miRNA effects are often studied in isolation, and it remains unclear how the repertoire of miRNAs work together to alter gene regulation in late-stage ovarian cancers. Future studies should investigate these differentially regulated exosome miRNAs individually and collectively to increase our understanding of how they contribute to gene regulation in resistant phenotypes.
Lastly, many chemorefractory components (hypoxia-induced) overlap with the aberrant factors that are present in chemoresistant secretomes (frontline drug-induced) ( Table 2). We believe that examining the unique circulating molecules in the chemorefractory secretome is imperative because HGSOC heavily relies on the secretome for metastasis. In fact, these factors support and maintain metastasizing HGSOC cells for subsequent secondary site attachment. Therefore, examining the factors that are present in this type of secretome can lead to new diagnostic measurements that can increase HGSOC detection.


Dielectrophoretic Traps for Efficient Bead and Cell Trapping and Formation of Aggregates of Controlled Size and Composition

We present a microfluidic dielectrophoretic-actuated system designed to trap chosen single-cell and form controlled cell aggregates. A novel method is proposed to characterize the efficiency of the dielectrophoretic trapping, considering the flow speed but also the heat generated by the traps as limiting criteria in cell-safe manipulation. Two original designs with different manufacturing processes are experimentally compared. The most efficient design is selected and the cell membrane integrity is monitored by fluorescence imaging to guarantee a safe-cell trapping. Design rules are suggested to adapt the traps to multiple-cells trapping and are experimentally validated as we formed aggregates of controlled size and composition with two different types of cells. We provide hereby a simple manufactured tool allowing the controlled manipulation of particles for the composition of multicellular assemblies.

INTRODUCTION
The necessity of developing tools able to manipulate single cells and analyse their behaviour at the single-object resolution is well established (Di Carlo et al., 2012;Dura et al., 2015;Abdulla et al., 2021). Unveiling cell heterogeneity by simultaneous temporal and single-cell resolution of cell response to external stimuli are difficult to obtain and often limited to one of the two with standard methods such as microscopy or flow cytometry. Microtechnologies offer the ability to manipulate objects at the single-cell resolution using different forces. In particular, dielectrophoresis (DEP) is a method of choice when a non-contact, active and versatile manipulation of cells or particles suspended in a liquid medium is required. Dielectrophoresis derives from a non-uniform electric field inducing a polarization of a particle. The direction of the force on the particle arising from this polarization is defined by design, but its magnitude can be tuned by the voltage applied to the electrodes and its positive (pDEP) or negative (nDEP) effect can be tuned in particular cases by the frequency. DEP has been widely used for separation of cells based on dielectric properties (Piacentini et al., 2011), but has also often been used to direct, trap and position single cells (Mittal et al., 2007;Godino et al., 2019;Punjiya et al., 2019). However, the presence of an electric field in a conductive medium can harm cells, and while many authors demonstrate the ability of their design to trap particles against a certain flow rate for a given voltage, very few assess the heat generation related to the traps (Seger-Sauli et al., 2005).
The formation of cell aggregates with controlled number and type of cells is crucial in the understanding of cancer invasion and development. For example the role of cancer associated fibroblasts in tumorigenicity is well known and the need for multicellular models based on co-culture to mimic the tumor environment was demonstrated (Labernadie et al., 2017;Lazzari et al., 2018), but there is a lack of tools to control the composition of multicellular assemblies down to the single cell level. In well plates, obtaining single cells using limiting dilution methods comes at the cost of a small portion only of usable wells due to Poisson distribution (Gross et al., 2015) and cell ratios are often determined based on volume and density to recreate heterogeneity (Bauleth- Ramos et al. (2020)). Dielectrophoresis has already been used as a tool to form aggregates of cells (Altomare et al., 2003;Menad et al., 2015;Cottet et al., 2019) and to trap and pair single cells in a controlled manner (Kirschbaum et al., 2008;Yoshimura et al., 2014) but has not yet, to our knowledge, been used to create heterogeneous aggregates with controlled number and type of cells.
In this work, we propose an original electrode design offering an efficient three-dimensional dielectrophoretic trap for single cells together with a distribution system. We propose two easy to fabricate configurations, compare their trapping efficiency and assess their heat generation. The most efficient configuration is selected and the limits of voltage necessary to avoid cell membrane electroporation is determined. The design rules to accommodate more cells in higher channels is defined. We demonstrate the capability of the presented system to direct and arrange cells in a controlled manner by forming multicellular assemblies of predetermined size and composition.
The device we propose is easy to fabricate and offers efficient three dimensional trapping capabilities with simple coplanar electrodes and is thus accessible to researchers disposing of standard equipment. The distribution system allows a full control on the positioning of the arriving cell and unlocks for the first time the ability to form heterogeneous assemblies of cells with pre-determined number and type of cells using DEP. This feature is key in research domains focusing on cancer stem cells and the corresponding drug development strategies (Ishiguro et al., 2017), but finds also applications in studies of cellular aggregates that mimic the cancer micro-environment (Ham et al. (2016)). While electroporation of the created assembly was avoided in this study, it can be exploited and is readily available in drug development or applications where cell transfection is desired (Chopinet et al. (2012)). Furthermore, domains studying cell adhesion and receptor-ligand interactions will benefit from this device's ability to form pairs of beads and cells (Dura et al. (2015)). We demonstrated here these features using an array of four traps, but the number of traps can be easily scaled up by lateral repetitions of the trap units as well as repetitions of pairs of electrodes along the length of the channel.
The facing electrodes chips were fabricated following a process described previously (Vulto et al., 2005), by first patterning electrodes as described in the previous paragraph ( Figure 1A steps 1a-1c). A photosensitive adhesive film was laminated (Ordyl ® SY320, 20 μm thickness) ( Figure 1B step 2), exposed through a mask using a mask aligner (MJB4, Süss) with a postbaking step of 1 min at 85°C, developed (Ordyl ® XFB) and FIGURE 1 | Process for the fabrication of the chips. (A) The fabrication of coplanar configuration chips starts by the patterning of Ti-Pt electrodes on a glass substrate (wafer 1). PDMS is cast on a silicon mold, punched, aligned and permanently bonded to the glass chips (wafer 2). (B) The fabrication of facing configuration chips starts by the patterning of electrodes on a glass wafer and is followed by the lamination and patterning of a photosensitive film to define the (wafer 1). The cap wafer coated with ITO (wafer 2) is then aligned and bonded to the bottom chip.
Frontiers in Bioengineering and Biotechnology | www.frontiersin.org July 2022 | Volume 10 | Article 910578 2 rinsed (Ordyl ® Rinse) ( Figure 1B step 3-4). The fluidic inlets and outlets of the capping borofloat wafer were fabricated by sandblasting (IcoFlex Sàrl, Switzerland) and 200 nm of ITO was then sputtered (SPIDER600, Pfeiffer) ( Figure 1B step 5). Both wafers were then aligned (MA6/BA6, Süss) and bonded (SB6, Süss) by applying a pressure of 425 kPa for 30 min at 90°C followed by a curing step at 150°C for 2 h ( Figure 1B step 6). Connections between the top and bottom electrode were made by placing a chip on a heating plate at 90°C and introducing low temperature solder (The Indium Corporation of America) inside a microfluidic channel designed for this application and passing on top of the desired bottom electrode track.

Materials
5 μm in diameter polystyrene beads were purchased from Sigma-Aldrich and suspended in a working solution composed of 10% in volume phosphate buffered saline (PBS) (Gibco) and 90% deionized water at a concentration of 5 · 10 5 beads/ml. The surface of polystyrene beads was left uncoated and the particles are thus natively negatively charged in buffers with physiological pH. The use of AC electric fields however discards any net movement due to electrostatic forces.

Cell Culture
Jurkat and Colo205 cell lines (ATCC) were cultured in RPMI 1640 supplemented with 10% of fetal bovine serum (FBS) and 1% Penicillin-Streptomycin at 37°C in 5% CO 2 atmosphere. Staining of Colo205 was performed by incubating the cells in PBS with 4 μm Calcein UltraBlue ™ AM (Cayman Chemical) for 1 h.

Staining of Jurkat cells was done by incubation in PBS with
1 μm Calcein AM (Invitrogen ™ ) for 1 h. The working solution is composed of 40% RPMI and 60% deionized water. The solution is compensated for osmolarity by the addition of dextrose (Sigma-Aldrich) and cleaned through a 0.22 μm filter. Jurkat and Colo205 were both resuspended in the working solution and passed through a 40 μm cell strainer before the experiment. All reagents are from Gibco unless specified.

Chip Operation
Measurement of the current and phase was performed in a solution of 10% PBS and 90% deionized water by applying an AC signal between 1 and 10 V amplitude to the trapping electrodes at a frequency of 100 kHz using an HF2TA current amplifier connected to a HF2LI Lock-In amplifier (Zurich Instruments).
The PDMS chip was primed with Pierce ™ Protein-Free (PBS) Blocking Buffer during 1 h to prevent cells from adhering to the surfaces. The cells or beads were placed in a chromatography vial connected to the punched PDMS by a 360 μm outer diameter PEEK tubing (Idex). Pressure was applied to the vials using Fluigent Flow-EZ pressure controllers. The chip was mounted on and electrically connected to a custom PCB placed on the stage of a Leica DMI3000 B inverted microscope and observed using a uEye (IDS) camera. All the electric signals needed to control the positions of the particles are sent through a home made PCB creating the multiplication of an AC signal at 100 kHz and different DC signals whose amplitudes are controlled by the computer with an adapted C++ program through an analog output generator (Mccdaq USB-3100).

COMSOL Simulations
Modeling of the electric field and DEP force direction and magnitude were done using COMSOL Multiphysics 5.6 with the Electric Currents and Creeping Flow modules. The medium electrical conductivity and relative permittivity were set respectively to 0.16 S/m and 78. The fluid flow at the entrance of the channel was set to 700 μm/s. A sinusoidal electric potential of 10 V amplitude and 100 kHz frequency was applied to the trapping electrode and the potential of the counter electrode was set to zero.

Concept
We propose a microfluidic system capable of performing in-flow cell selection, sorting and trapping using non-contact DEP actuation. The system is presented in Figure 2A is composed of an upstream DEP actuated deviation system that laterally focuses the incoming particles or cells on specific flow lines so that they can be collected by the desired downstream DEP trap. The DEP traps are actuated by applying a potential across two electrodes: the upstream main electrode is designed with repetitions of funnel-shaped trap units and the counter electrode is either placed in a coplanar or facing configuration. Each trap unit is 60 μm in width and capable of stopping a single cell or particle in flow by balancing the drag force and creating a single position of equilibrium upstream of the electrodes. The slanted parts of the trap unit finely focus the particles towards the center of the traps indicated by the focus line in Figure 2A. The number of traps can be adjusted to the width of the channel by lateral repetitions of the funnel-shaped trap unit. A bypass area is also provided next to the trapping electrodes such that unwanted cells can be directed to bypass the traps and leave the chamber.

Deviation System
In order to control the trajectory of the incoming particles and guide them towards the desired trap unit, a deviation system using six pairs of liquid electrodes similar to the one presented by Demierre et al. (2007) was placed upstream of the trapping chamber. Only two pairs of liquid electrodes are represented on the left of Figure 2A. Liquid electrodes are coplanar electrodes placed in a lateral recess from the main channel: this configuration guides the electric field and generate a vertical equipotential surface at the entrance of the main channel, hence the name "liquid electrodes". Two sets of liquid electrodes combs are placed laterally on each side of the main channel and a voltage is applied to each set, with a phase shift of 180°to neighbouring electrodes. Each set of liquid electrodes generate a lateral DEP force uniform along the height of the channel pushing the particles away from them. The ratio of voltage applied to each side V 1 /V 2 determines the equilibrium position along the y axis in the main channel, focusing the randomly distributed incoming particles towards a defined lateral position and deviating them Frontiers in Bioengineering and Biotechnology | www.frontiersin.org July 2022 | Volume 10 | Article 910578 towards the trap units, or to the bypass. As described by Demierre et al. (2007), the total force exerted on the particles is the sum of the forces exerted by each comb of electrodes and the particles are focused where these forces cancel each other: it is thus non dependent on the particle or cell size nor position before entering the deviation zone. At low fluid velocity, the limit in the focusing is due to particles collision and diffusion, which is compensated by the slanted part of the traps placed downstream, collecting particle streams covering 60 μm in width. The focusing nevertheless has a size dependent limit on the channel sides: the particle's center cannot be focused closer to the wall than the particle's radius due to straightforward reasons.

Counter Electrode Configuration and Simulations
Two easy to fabricate configurations of counter electrodes were implemented and studied: a coplanar counter electrode which consists in a straight line electrode placed downstream of the trapping electrode as shown in Figure 2B, whereas for the facing counter-electrode, a conductive and transparent ITO layer is provided on the glass plate used to close the channels on their upper part as illustrated in Figure 2C. Both configurations are popular and extensively used to manipulate cells using DEP in microfluidic chips (Ho et al. (2013); Li and Bashir (2002); Morgan et al. (1999); Takahashi and Miyata (2020)). Other electrodes configurations that are known to create single cell traps comprise 3D electrodes (Keim et al., 2019) and strip electrodes (Seger et al., 2004), but such configurations are less accessible in terms of fabrication. In order to assess the three dimensional behaviour of the traps, both designs were simulated using COMSOL ( ™ ) .   Figure 2A) for both configurations and for a voltage amplitude of 10 V. The colour indicates the magnitude of zE 2 zx , which is the electric field dependent factor that modulates the DEP force, while the arrows and streamlines indicate the direction of the nDEP force along the xz plane. The electrodes are drawn on top of the simulations for visualization. Both electrodes configuration generate a force pushing against the flow and towards the bottom of the channel upstream of the main electrode. As the y component of the force points towards the center of a trapping unit thanks to the slanted part of the electrodes (see Supplementary Figure S1), both configurations generate a three dimensional trap against the flow.
Frontiers in Bioengineering and Biotechnology | www.frontiersin.org July 2022 | Volume 10 | Article 910578 Figure 3 illustrates the results of the simulation along the focus line indicated in Figure 2A: To test these two designs, chips with electrodes placed in both configurations and aligned to microfluidic channels with μm precision can be manufactured using standard cleanroom equipment with a few steps only. The fabrication process for both designs is detailed in the Materials and Methods section and illustrated in Figures 1A,B. Experiments are presented in the next section.

Bead Trapping Efficiency
DEP can damage cells as it can induce a local rise in temperature. Indeed the presence of an electric field in a conductive media induces Joule heating. This effect can be mitigated by reducing the conductivity of the medium and correcting for osmolarity with the addition of dextrose or sucrose, but prolonged exposition of cells to such diluted media can alter their function and health (Hyler et al. (2021)). As both electrodes configurations presented here create a three dimensional DEP trap against the flow, we selected the most efficient configuration by measuring the magnitude of the DEP force against the flow for both configurations and evaluated in both cases the induced temperature rise. The expression of the time averaged DEP force exerted on a spherical particle in a non uniform electric field is given as follows: Where R is the radius of the particle, ϵ m is the fluid permittivity, Re [K(ω)] is the real part of the Clausius-Mossotti factor and E rms is the root mean square (rms) of the electric field. For a homogeneous spherical particle, the Clausius-Mossotti factor is given by the following formula: With ϵ p p the complex permittivity of the particle and ϵ p m the complex permittivity of the medium, which are both frequency dependent. The sign of the Clausius-Mossotti defines the regime of the DEP force, negative or positive, resulting in particles being respectively repulsed from or attracted to the high electric field regions. In this work, we operate at 100 kHz in the negative DEP regime and the real part of the Clausius-Mossotti factor has a value of −0.5 for all medium and particle or cell conditions.In order for a particle to be trapped, the projection of the DEP force on the axis of the flow direction (x in the present case) has to balance the viscous drag force exerted by the flow (Voldman et al. (2001); Rosenthal and Voldman (2005)): Where η is the fluid viscosity, v mean is the mean fluid velocity, h is the height of the channel, F* is a factor accounting for the wall effect, z p is the height of the particle. The heat flow generated by electrodes matches the electrical power input in the system and the average temperature around the DEP electrodes was shown to depend on the real part of the electrical power (Ramos et al., 1998;Seger-Sauli et al., 2005) which is defined as: With V rms the rms voltage applied to the electrodes, I rms the rms electrical current, θ the phase shift between the current and voltage and Z the norm of the electrical impedance.
Since the temperature is directly proportional to the electrical power, this latter can be used as an indicator of electrodes trapping efficiency when comparing designs of similar size. Following Equation (3), the DEP force developed by a trap at steady state and at a defined position is directly proportional to the velocity of the fluid flow dragging the particle. We thus measured the maximum DEP force developed by each configuration by immobilizing a polystyrene particle in a trap at low fluid flow and increasing the flow until the bead is released. We measured the particle velocity at release, which is directly proportional to the maximum DEP force the trap can develop in the x direction at the edge of the electrode as a function of applied voltage and developed power. This latter is obtained by multiplying the applied voltage by the measured current at each condition following Eq. 4. From Eq. 3, the mean velocity of the fluid can be expressed as: Since the potential distribution in space is a direct function of the voltage V applied to the electrodes, we can derive that (see Supplementary Information) ∇E 2 rms α(x, y, z)V 2 , where α is a function of space. Eq. 5 can thus be re-written as: Introducing the electrical power into this equation from Eq. 4 we obtain: Frontiers in Bioengineering and Biotechnology | www.frontiersin.org July 2022 | Volume 10 | Article 910578 With α x the x component of α(x, y, z) described above and taken at the edge of the electrode, this equation indicates that the efficiency of trapping of a given design and configuration of electrodes depends on the multiplication of α x and Z.
The current and phase were measured for both configurations and for voltage amplitudes between 1 and 10 V. The real part of the electrical power, responsible for Joule heating, was calculated as of Eq. 4. The mean and standard deviation phase was measured to be 11.2°and 1.4°respectively for the coplanar configuration and 8.9°and 1.5°respectively for the facing configuration, the real part of the power accounted for more than 97% of the apparent power thus indicating a mainly resistive load. Figure 4A is a plot of the measured speed at release for 5 μm in diameter polystyrene particles as a function of applied voltage amplitude for both facing and coplanar configurations. The dependency of the velocity at release is quadratic on the voltage in accordance with Eq. 6. As predicted by the simulations and Figure 3, the facing configuration generates a larger zE 2 rms zx for a given voltage and the velocity of the particle at release is larger in this configuration. Figure 4B shows the velocity at release as a function of electric active power. It results that the velocity at release depends linearly on the power as predicted from Eq. 7. It appears that the larger gradient factor α x of the facing design does not compensate the smaller impedance of this design, resulting in more heat dissipated for a given DEP force than the coplanar design and thus a larger increase in temperature. The coplanar design was thus selected for the following study of cell trapping. However, the facing electrode configuration is suggested for applications where flow speed is the main criteria and heat generation is not a limitation since it generates a larger DEP force for a given voltage.

Single Cell Trapping
Electroporation can damage the cells when trapped using DEP. Electroporation takes place when the potential difference accross the cell membrane exceeds a threshold value, inducing pores in the membrane. This phenomenon is not necessarily lethal for the cells and is widely exploited to introduce genetic material inside the cells. However electroporation is not desired in manipulation applications and to avoid any damage to the cells we experimentally determined the applied voltage limit to avoid electroporation conditions. Jurkat cells were loaded with Calcein AM to visualize pore formation: as calcein is a volatile fluorescent molecule, it quickly diffuses out of the cell in case of pore formation in the membrane. The maximum velocity for trapping without electroporation as a function of voltage amplitude is measured and reported in Figure 5A. In case of fluorescence loss, the voltage was immediately turned off to release the cell and measure its speed. Events where a leak of calcein was observed are indicated by an orange circle, whereas events without fluorescence loss are indicated by a blue cross. A clear threshold under which no fluorescence loss is observed is found for a voltage of 5 V amplitude and determines the voltage operation limits to avoid cell damage. The large variation in speed at release can be explained by variations in cell size. Figure 5B is a picture of a cell arriving in the trap and Figure 5C shows the cell after fluorescence loss.
Typical transmembrane potential threshold above which pores appear have been reported between 0.25 and 1 V (Escoffre et al. (2011)). Schwan equation relates the transmembrane potential ΔΦ m to the external alternating electric field E with angular frequency ω: Given τ = RC mem (ρ int + ρ ext /2) with C mem the membrane capacitance, ρ int and ρ ext the resistivity of respectively the internal and external fluid and φ the angle between the electric field lines and a line drawn from the center of the cell to the considered point of interest on the cell membrane. The critical value for transmembrane potential corresponds to a range of electric field between 2.8·10 4 and 11.2·10 4 V/m using membrane properties values from Reichle et al. (2000). Such values of electric field are found at the edge of the electrode and for a height of 5 μm in simulations for applied voltage amplitude ranging between 2.3 and 9.3 V and comprising the experimentally FIGURE 4 | Characterization of the trapping efficiency: 5 μm in diameter polystyrene beads are trapped for a given voltage and power and the flow is increased until the bead takes off and its speed is measured. The black dots and bars are the mean and standard deviation respectively and the continuous line is the fit. (A) The speed at release follows a squared dependency on the applied voltage as predicted by Eq. 6. As expected from simulations in Figure 3, the coplanar configuration develops a smaller force for a given applied voltage compared to the facing configuration. (B) The speed at release depends linearly on the developed power as expected from Eq. 7. It can be deduced that the coplanar design is more efficient at trapping in this case as it can develop a larger F DEP,x for a given power dissipated than the facing configuration.
Frontiers in Bioengineering and Biotechnology | www.frontiersin.org July 2022 | Volume 10 | Article 910578 6 found threshold, consolidating the hypothesis of fluorescence leakage due to electroporation. The maximum voltage for cells manipulation without electroporation was thus set to 4 V amplitude. Larger voltages may be used without electroporating the cells as long as the flow drag force is limited and does not bring the cell in the critical electric field close to the electrode edge.

Multiple Cells Trapping
The presented cell DEP traps with controlled deviation system can be used to trap multiple particles of different types in a single trap. Indeed the formation of aggregate of controlled composition and size can be of high interest to study the growth of tumors in their environment composed of different cell types. For applications that require to stop more than 1 cell, the trap and microfluidic channel need to be scaled up.

Scaling Rules
Different parameters were studied to understand their impact on the three-dimensional trapping property for an increased channel height. We defined the following parameters: d is the distance between the electrodes, l is the depth of the electrodes, h is the height of the channel and e is the width of the parallel part of the main electrode as illustrated on the inset of Figure 6. The angle of    (Rosenthal and Voldman (2005)). Indeed, particles with a center of mass in the region with upward DEP force will be pushed upwards where the DEP force counteracting the flow is weaker, and therefore leave the trap. It is especially a problem when trapping multiple objects where the chance of finding an object higher in the channel is larger. As shown in the previous discussion, the standard design has a vertical contour and all particles coming in the traps will be pushed down to the floor. However, Figure 6B shows that this property is lost when keeping the same electrode geometry and increasing the channels height h. The scaling of the electrodes depth l and distance between them d is not enough to recover the vertical contour as seen on Figure 6C. A homothetic scaling is necessary to obtain the property of DEP force pushing down to a single equilibrium position along the whole trap height and to create a compact aggregate, as shown in Figure 6D.

Formation of Aggregates of Controlled Size and Composition
We demonstrate here the ability of the presented system to create cell aggregates of controlled size and composition. The single cell design was scaled up as in Figure 6D to accommodate more cells in a channel height of 40 μm. Four parallel trapping units were placed next to each other and a bypass with no electrode was left next to the trapping units in order to discard unwanted cells. Two inlets were used sequentially to perfuse solutions with respectively Colo205 cells stained with blue calcein AM, and Jurkat cells stained with green calcein AM. The upstream deviation system was used to direct the incoming cells toward the desired trapping unit to create aggregates of 4 cells composed of three Colo205 and one Jurkat (Figures 7A-C) as well as two Colo205 and two Jurkat ( Figures 7D-F). The calcein staining was used to both identify the cell type as well as to ensure cell viability. The aggregates could be held inside the traps up to 5 minutes without witnessing any leakage of the dye indicating membrane poration.

CONCLUSION
This study first proposes a design of electrodes capable of generating three-dimensional single-object DEP traps in two different configurations of counter electrodes. This design offers the possibility of increasing the number of traps actuated simultaneously and we demonstrate the use of a DEP deviation system to direct the particles towards the desired traps in a controlled fashion. We proposed a simple method for the evaluation of DEP traps efficiency for designs comparison. The method evaluates the maximum DEP force the trap is capable of developing to counteract the drag force and assesses its relationship to the power, mainly dissipated in Joule heating, necessary to generate it. We used this method to compare the two Frontiers in Bioengineering and Biotechnology | www.frontiersin.org July 2022 | Volume 10 | Article 910578 8 types of electrodes configurations and deduce that coplanar electrodes configuration is more efficient than the facing electrodes configuration. The coplanar configuration was used to trap Jurkat cells and the voltage limit to avoid electroporation was experimentally assessed. The scaling rules were defined to follow changes in channel height and a scaled design was used to create aggregates of 4 cells with controlled number and type of cells. We defined the voltage threshold for electroporation events, and when performed in a controlled way, a pulse of high voltage once cells are trapped can offer the possibility of controlled electroporation on specific cells or aggregates for transfection or electrofusion applications.
We believe that the proposed method to evaluate the efficiency of DEP traps based on power dissipation is crucial when trapping cells. The resulting coplanar configuration chip with deviation system offers a versatile tool for single cells and cell aggregates manipulation and studies and we believe will be useful to study biological interactions between cells and cellular assemblies.
Future development of the device will comprise a scale up in the number of traps and the full automation of the system using computer vision or impedance based feedback. Additional deviation system at the outlet of the interaction chamber will enable the sorting of the created assemblies. Additionally, placing a chamber comprising an array of hydrodynamic traps such as those presented by Bourn et al. (2020) to immobilise the formed assemblies downstream of the interaction chamber will allow a change of medium for long term on-chip culture and their longitudinal studies.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

FUNDING
This work was supported in part by the EIPHI Graduate School under Contract ANR-17-EURE-0002 and in part by the French Agence Nationale de la Recherche and the Swiss National Science Foundation through the CoDiCell project (contract "ANR-17-CE33-0009" and "No. 00021E_175,592/1", respectively). Open access funding provided by École Polytechnique Fédérale de Lausanne.