Nicolas Sahakian

Frederic Castinetti

Henry Dufour

Thomas Graillon

Pauline Romanet

Anne A Barlier

Thierry Brue

Thomas Cuny

Clinical management of difficult to treat macroprolactinomas

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

General considerations on prolactinomas

Prolactinomas represent the most common hormone-secreting pituitary tumor encountered in the clinic with a prevalence of 100 per million of the population [START_REF] Gillam | Advances in the treatment of prolactinomas[END_REF][START_REF] Colao | The prolactinoma[END_REF]. It currently accounts for 30-40% of all pituitary adenomas (PA) and, depending on their maximal diameter, they are commonly classified into microprolactinomas (<10 mm) or macroprolactinomas (≥ 10 mm). Histopathological data previously learned us that prolactin (PRL) can be secreted by several types of PiTNETs deriving either from lactotrophs, somatotrophs or mammosomatotrophs cells. Likewise, sparsely or densely granulated lactotroph tumors, acidophil stem cell tumors, poorly differentiated Pit1-lineage tumors can also lead to hyperprolactinemia. Therefore, in the following sections we will exclusively refer to the most common type of PRLsecreting PiTNETs, namely the one arising from lactotroph cells and called as Prolactinomas thereafter. Prolactinomas are overwhelmingly diagnosed in women population with a sex ratio estimated to be 10:1 before the fifth decade [START_REF] Gruppetta | Prevalence and incidence of pituitary adenomas: a population based study in Malta[END_REF]. After this age, the frequency of Prolactinomas is the same in both gender, although macroprolactinomas are generally diagnosed at a later age in men as compared to women (median age incidence of 51 years in men vs. 34 to 42 years in women). In children and adolescents, PA is a rare pathological condition (less than 2% of all intracranial tumors) and prolactinomas represent about half of the PA phenotypes encountered in this population [START_REF] Ciccarelli | The epidemiology of prolactinomas[END_REF].

Because of their remarkable response-to-treatment, their slowgrowing behavior and their almost non-existent propensity to metastasize, Prolactinomas represent the archetype of benign tumors. In few cases however, they can present with stigmas of aggressivity represented by either invasion of perisellar structures (e.g. the cavernous sinus) and/or several relapse in spite of a wellconducted treatment. Accordingly, pathologists and clinicians adopted the terminology Pituitary Neuroendocrine Tumors (PiTNETs) instead of PA [START_REF] Asa | From pituitary adenoma to pituitary neuroendocrine tumor (PitNET): an international pituitary pathology club proposal[END_REF].

In exceptional cases, the primitive PRL-secreting tumor can be complicated by releasing of tumoral cells in the cerebrospinal fluid and/or distant metastasis in the central nervous system, two conditions that defined a PRL-secreting carcinoma [START_REF] Heaney | Pituitary carcinoma: difficult diagnosis and treatment[END_REF].

The clinical symptoms and complains from patients diagnosed with microprolactinomas are usually the consequence of the hyperprolactinemia-induced gonadotroph deficiency, namely irregular menstruation or amenorrhea, decreased libido and erectile dysfunction. These symptoms reflect the inhibitory effect exerted by PRL on the reproductive axis by reducing both the secretion and pulsatility of gonadotropin-releasing hormone (GnRH) neurons from the hypothalamus [START_REF] Abbara | Interpretation of serum gonadotropin levels in hyperprolactinaemia[END_REF][START_REF] Page-Wilson | prolactin suppresses GnRH but not TSH secretion[END_REF]. The latter effect could be mediated through inhibition of Kisspeptin neurons, which express PRL receptors and are major regulators of GnRH neurons [START_REF] Sonigo | Hyperprolactinemia-induced ovarian acyclicity is reversed by kisspeptin administration[END_REF][START_REF] Kaiser | Hyperprolactinemia and infertility: new insights[END_REF]. The involvement of Kisspeptin neurons have been also demonstrated in both sexual and emotional functions which could, at least partially, explain the decreased libido observed in patients with hyperprolactinemia [START_REF] Comninos | Modulations of human resting brain connectivity by kisspeptin enhance sexual and emotional functions[END_REF]. Besides irregular menstruations, PRL-secreting PiTNETs can cause galactorrhea and be associated with an increased risk of vertebral fractures in both men and women [START_REF] Mazziotti | Vertebral fractures in males with prolactinoma[END_REF][START_REF] Mazziotti | High prevalence of radiological vertebral fractures in women with prolactin-secreting pituitary adenomas[END_REF].

In macroprolactinomas, symptoms at the diagnosis include headaches, visual field impairment, symptoms related to anterior pituitary deficiencies and/or hyperprolactinemia [START_REF] Tirosh | Hypopituitarism patterns and prevalence among men with macroprolactinomas[END_REF].

Accordingly, the aim of the therapy in patients with macroprolactinomas is therefore twofold:

(1) to reduce and ideally normalize the serum PRL levels.

(2) to control the tumor volume, with recovery of the visual defect and/or pituitary functions when deficient.

The aim of this review is to summarize the current and recent therapeutical strategies to optimize the clinical management of difficult-to-treat macroprolactinomas.

The medical treatment of prolactinomas

In patients with Prolactinomas, we previously assumed that the aim of the therapy is to restore normal gonadal functions, fertility and to reduce tumor size [START_REF] Molitch | Diagnosis and treatment of pituitary adenomas[END_REF]. DAs represent the cornerstone of the medical treatment and are always discussed as the first-line therapy in micro-as well in macroprolactinomas [START_REF] Tirosh | Management of macroprolactinomas[END_REF]. Their action is based on the property the lactotroph adenoma cells has to express high level of the dopamine receptor subtype 2 (D2DR) which, once binded by DA, led to the inhibition of both PRL secretion and cell proliferation [START_REF] Ben-Jonathan | Dopamine as a prolactin (PRL) inhibitor[END_REF]. Three DA are currently available, two that are ergot-derived compounds (i.e cabergoline and bromocriptine), and one, quinagolide, which is a non-ergotderived DA [START_REF] Cuny | Medical therapies in pituitary adenomas: current rationale for the use and future perspectives[END_REF]. All of these DA are orally administered and their efficacy in the treatment of macroprolactinomas or in case of primary resistance to DA is briefly resumed in Table 1.

Bromocriptine

Bromocriptine (BRC) is the first DA which were used for the treatment of Prolactinomas as its prescription started at the end of the 80's [START_REF] Molitch | Bromocriptine as primary therapy for prolactin-secreting macroadenomas: results of a prospective multicenter study[END_REF][START_REF] Dallabonzana | Chronic treatment of pathological hyperprolactinemia and acromegaly with the new ergot derivative terguride[END_REF]. A posology ranging from 2.5 mg to 15 mg per day (median of 7.5 mg per day) is commonly used but higher doses until 20 to 30 mg per day can be required in almost a third of patients [START_REF] Colao | Medical treatment of prolactinomas[END_REF]. Results of a plethora of studies indicate that for microprolactinomas, BRC succeeds to normalize PRL levels, restores gonadal functions and decreases tumor mass in 80-90% of patients, while similar outcomes are found in 70% of patients with macroprolactinomas [START_REF] Colao | Medical treatment of prolactinomas[END_REF]. One of the major issues of BRC is the occurrence of adverse side effects, which can be observed in up to 10-12% of patients and mainly represented by digestive (nausea/vomiting, constipation, reflux, pain), neurological (headaches, dizziness, confusion, dyskinesia) and cardiovascular (postural hypotension, syncope) symptoms [START_REF] Wong | Update on prolactinomas. Part 2: treatment and management strategies[END_REF][START_REF] Kissner | Side effects of bromocriptine[END_REF]. They generally occur after the initial dose and with dosage increases, but can be minimized by introducing the drug at a low dosage (0.625 or 1.25 mg/d) at bedtime, by taking it with food, and by very gradual dose escalation [START_REF] Gillam | Advances in the treatment of prolactinomas[END_REF]. BRC has been largely superseded by other DA with longer lasting effects and improved side effect profiles.

Even its historical preferential use over others DA during pregnancy is now null [START_REF] Molitch | Endocrinology in pregnancy: management of the pregnant patient with a prolactinoma[END_REF].

Cabergoline

Cabergoline (CAB) is a selective D2DR agonist which currently represents the most efficient DA as compared to BRC and quinagolide [START_REF] Webster | A comparison of cabergoline and bromocriptine in the treatment of hyperprolactinemic amenorrhea. cabergoline comparative study group[END_REF][START_REF] Colao | Drug insight: cabergoline and bromocriptine in the treatment of hyperprolactinemia in men and women[END_REF]. In a retrospective study assessing 455 cases of hyperprolactinemia, amongst who 41% and 42% were due to PRL-secreting micro-and macroadenomas, respectively, the PRL excess had been normalized in a total of 86% of cases under cabergoline treatment [START_REF] Verhelst | Cabergoline in the treatment of hyperprolactinemia: a study in 455 patients[END_REF]. More specifically, the normalization of PRL level was obtained in 77% of patients with macroprolactinomas [START_REF] Verhelst | Cabergoline in the treatment of hyperprolactinemia: a study in 455 patients[END_REF]. Similar results were observed in successive studies, which, in parallel, demonstrated that CAB restored menses in 82% of women with amenorrhea [START_REF] Webster | A comparison of cabergoline and bromocriptine in the treatment of hyperprolactinemic amenorrhea. cabergoline comparative study group[END_REF], improved fertility by normalizing the sperm quality and sexual function in male [START_REF] Colao | Outcome of cabergoline treatment in men with prolactinoma: effects of a 24-month treatment on prolactin levels, tumor mass, recovery of pituitary function, and semen analysis[END_REF] and eventually led to a significant tumor shrinkage in around 90% of patients with Prolactinomas [START_REF] Colao | macroprolactinoma shrinkage during cabergoline treatment is greater in naive patients than in patients pretreated with other dopamine agonists: a prospective study in 110 patients[END_REF]. Male gender, invasive growth and giant tumors (i.e. diameter >4 cm) are associated with a lower response to cabergoline [START_REF] Delgrange | Characterization of resistance to the prolactin-lowering effects of cabergoline in macroprolactinomas: a study in 122 patients[END_REF][START_REF] Delgrange | Giant prolactinomas in women[END_REF][START_REF] Salenave | Macroprolactinomas in children and adolescents: factors associated with the response to treatment in 77 patients[END_REF]. A starting posology of 0.5 mg/week is common, including in cases of macroprolactinomas, as the antitumor effect observed with this compound can be both massive and rapid, as exemplified by the occurrence of chiasmal herniation (and visual field impairment) that have been described with low posologies of CAB [START_REF] Raverot | Secondary deterioration of visual field during cabergoline treatment for macroprolactinoma[END_REF].

Similarly, cases of cerebrospinal fluid rhinorrhea were described in the weeks following the initiation of CAB when the macroprolactinoma invaded the sphenoidal sinus [START_REF] Netea-Maier | Cerebrospinal fluid leakage as complication of treatment with cabergoline for macroprolactinomas[END_REF]. If low doses of CAB do not normalize hyperprolactinemia and if the patient tolerance is good, it is worthwhile to progressively increase the dose of the drug. As such, around 60% of patients with macroprolactinomas will achieve a normalization of their hyperprolactinemia under CAB in an average delay of less than two years [START_REF] Paepegaey | Cabergoline tapering is almost always successful in patients with macroprolactinomas[END_REF]. In a monocentric study, 44 patients (19.6%) received CAB doses >2 mg/week with an average interval of 8 months between each dose increment, and a definitive CAB resistance diagnosed in half of the patients (21 had a partial response to CAB with decrease but no normalization of PRL level and 1 was unresponsive at all) [START_REF] Paepegaey | Cabergoline tapering is almost always successful in patients with macroprolactinomas[END_REF]. Thus, increasing the dose of CAB with periodical monitoring of both PRL levels and tumor volume seems reasonable over a period of 24 months, before stating that the tumor is resistant. Whether increasing the posology of CAB can expose the patient to develop cardiac valvulopathy was still source of uncertainty. However, a recent study showed that for the posologies generally used in the treatment of Prolactinomas, the use of CAB was safe and did not overexpose the patient to the risk of valvulopathy [START_REF] Vroonen | Prospective, long-term study of the effect of cabergoline on valvular status in patients with prolactinoma and idiopathic hyperprolactinemia[END_REF].

In conclusion, CAB is usually the first-line DA to be proposed with a side effects profile similar to those reported for the other DAs, but generally less frequent, less severe, and of shorter duration [START_REF] Melmed | Diagnosis and treatment of hyperprolactinemia: an endocrine society clinical practice guideline[END_REF]. The only serious shortcoming currently under active investigation is the occurrence of a compulsive behavior in 5% of cases, such as excessive gambling and hypersexuality [START_REF] Noronha | Treating prolactinomas with dopamine agonists: always worth the gamble?[END_REF].

Quinagolide

Quinagolide (QNA) is a non-ergot-derived D2DR selective agonist which led, when administrated once-daily in patients with macroprolactinomas, to a significant reduction of tumor size and PRL levels in around 90% of patients and a further normalization of PRL level in 50% of them [START_REF] Van't Verlaat | Treatment of macroprolactinomas with a new non-ergot, long-acting dopaminergic drug, CV 205-502[END_REF][START_REF] Kvistborg | Long-term treatment of macroprolactinomas with CV 205-502[END_REF][START_REF] Schultz | Quinagolide in the management of prolactinoma[END_REF][START_REF] Sarno | The effect of quinagolide and cabergoline, two selective dopamine receptor type 2 agonists, in the treatment of prolactinomas[END_REF]. It is usually administered at the average dose of 150 to 300 μg per day. As compared to treatment with BRC, a greater reduction in nausea, vomiting, dizziness, and drowsiness during QNA administration is generally observed.

Other dopamine agonists

Pergolide is an ergot-derivated DA with long-acting D1 and D2 agonist properties, 100 times more potent than BRC allowing effective control of hyperprolactinemia with once-daily dose. However, it has been withdrawn from the market in 2007 because of cardiac valves adverse effects [START_REF] Schade | Dopamine agonists and the risk of cardiac-valve regurgitation[END_REF][START_REF] Zanettini | Valvular heart disease and the use of dopamine agonists for Parkinson's disease[END_REF] Lisuride is another ergot-derivated DA which was assessed for its ability to inhibit PRL secretion [START_REF] Colao | Medical treatment of prolactinomas[END_REF]. However, it never reaches the development to be used as a medical treatment in PRL-secreting adenomas. Finally, terguride, an analog of lisuride, can normalize PRL levels and reduce tumor size in a few number of patients with microprolactinomas, however it is currently not commercialized [START_REF] Dallabonzana | Chronic treatment of pathological hyperprolactinemia and acromegaly with the new ergot derivative terguride[END_REF].

Prolactinomas resistant to dopamine agonists

The resistance-to-DA therapy represents the most challenging condition of difficult-to-treat macroprolactinomas. It systematically requires a multidisciplinary approach to optimize the care of the patient.

Definition of the resistance

The definition of resistance-to-DA still constitutes a source of uncertainty, and multiple definitions have been proposed in the literature. One, which is recurrent, defines the resistance as failure to achieve normal serum PRL levels under DA (commonly admitted CAB ≥ 2 mg/week) or failure to reduce tumor size by at least 50% from the initial volume [START_REF] Molitch | Management of medically refractory prolactinoma[END_REF] (Figure 1). The cut-off of 2 mg/week chosen on purpose for CAB resistance actually makes sense. Indeed, in the group of patients treated with more than ≥ 2 mg/week of CAB, only a third of them will normalize their PRL level under 3 mg/week [START_REF] Ono | Prospective study of high-dose cabergoline treatment of prolactinomas in 150 patients[END_REF]. Therefore, the concept of resistance-to-DA does not mean per se that it is impossible to normalize PRL secretion but rather refers to a state in which the usual posologies of DAs do not achieve None.

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normalization of PRL levels or significant decrease of the tumor volume. According to these criteria, the prevalence of DAresistance in patients with Prolactinomas is 10 to 15% [START_REF] Vroonen | Prolactinomas resistant to standard doses of cabergoline: a multicenter study of 92 patients[END_REF] .

Molecular mechanisms underlying the resistance to DA

3.2.1. Alterations in the dopamine receptor subtype 2 (D2DR)

Most of the studies that focused on the resistance-to-DA in Prolactinomas investigated the pattern of D2DR expression both at the mRNA and at the protein levels (Figure 2). Our group showed a significant decrease of the D2DR mRNA transcription and identified alterations in the D2DR-related signaling pathways of BRC-resistant human Prolactinomas, as compared to sensitive tumors [START_REF] Pellegrini | Resistance to bromocriptine in prolactinomas[END_REF][START_REF] Pellegrini | Abnormal dopamine sensitivity in some human prolactinomas[END_REF][START_REF] Caccavelli | Decreased expression of the two D 2 dopamine receptor isoforms in bromocriptine-resistant prolactinomas[END_REF][START_REF] Caccavelli | Alteration of G alpha subunits mRNA levels in bromocriptine resistant prolactinomas[END_REF]. More recently, the differential expression of D2DR isoforms was proposed as a putative molecular mechanism which leads to different response to DA. In the study conducted by Shimazu et al., patients with surgically resected Prolactinomas were divided, based on their response to DA, in responders (n = 5), resistants (n = 5) and secondary resistants to DA (n = 2). The authors found a significant decrease of the D2DR long isoform mRNA expression in resistant and secondary resistant tumors as compared to the sensitive ones [START_REF] Shimazu | Resistance to dopamine agonists in prolactinoma is correlated with reduction of dopamine D2 receptor long isoform mRNA levels[END_REF]. In a similar approach, the correlation between the D2DR gene polymorphisms and the response to CAB was studied in a series of 148 patients with Prolactinomas (among whom 29 males and 75 macroadenomas), however without significant results [START_REF] Bueno | Cabergoline and prolactinomas: lack of association between DRD2 polymorphisms and response to treatment[END_REF].

A trend for similar results was found in the study of Filopanti et al. where the allele frequencies (i.e. the TaqI-A, TaqI-B, HphI and NcoI alleles) of the four D2DR polymorphisms was compared between sensitive-and resistant-to-DA Prolactinomas, without any significant correlation eventually observed [START_REF] Filopanti | Dopamine D2 receptor gene polymorphisms and response to cabergoline therapy in patients with prolactin-secreting pituitary adenomas[END_REF]. Nonetheless, in that study, a higher frequency of the NcoI-T allele was observed in resistant as compared to sensitive patients, which could suggest that this variant may lead to reduction of the expression and instability of the D2DR [START_REF] Filopanti | Dopamine D2 receptor gene polymorphisms and response to cabergoline therapy in patients with prolactin-secreting pituitary adenomas[END_REF]. Recently, the wholeexome sequencing analysis of 12 human Prolactinomas (Six considered as responsive and six as resistant under a dose of 15 mg/day of BRC, 11/12 patients with macroprolactinomas) revealed a differential expression of the PRDM2 (PR domain zinc finger protein 2) gene, about five-fold lower in resistant tumors as compared to the responsive ones [START_REF] Gao | Lower PRDM2 expression is associated with dopamine-agonist resistance and tumor recurrence in prolactinomas[END_REF]. The PRDM2 gene encodes a protein whose major role is to stabilize chromosomal structures, mediates gene expression and ultimately plays a role of tumor suppressor gene [START_REF] Cheedipudi | A fine balance: epigenetic control of cellular quiescence by the tumor suppressor PRDM2/ RIZ at a bivalent domain in the cyclin a gene[END_REF]. In resistant-to-DA Prolactinomas, the expression level of the PRDM2 protein was also significantly decreased as compared to sensitive cases and its overexpression in the MMQ cells, a rat PRL-secreting pituitary tumor cell line, led to the upregulation of D2DR expression and potentiated the inhibitory effect of BRC over PRL secretion [START_REF] Gao | Lower PRDM2 expression is associated with dopamine-agonist resistance and tumor recurrence in prolactinomas[END_REF].

The TGF beta pathway

Amongst its multifunctional known roles as a cytokine, TGF beta (TGF-β) can also regulates the proliferation of lactotroph cells as well as their PRL secretion [START_REF] Recouvreux | The pituitary TGFβ1 system as a novel target for the treatment of resistant prolactinomas[END_REF]. The TGF-β signaling cascade is initiated by the binding of TGF-β1, TGF-β2, and TGF-β3 ligands to the type II TGF-β receptor (TGF-β RII), followed by recruitment b.

a.

Figure 1. MRI aspect of a DA-resistant macroprolactinoma in a 33-year-old patient.

[A] Before cabergoline (CAB) introduction, the patient was diagnosed with left diplopia and erectile dysfunction. Hormonal analysis revealed a prolactin (PRL) level up to 4800 ng/mL. [B] MRI after 12 months of CAB therapy. Posology increased gradually from 0.5 to 3 mg/week at the last follow-up. PRL decreased to 1200 ng/mL, but never reached the normal range and tumor volume shrinkage was considered to be less than 50% from the initial tumor volume. Interestingly, diplopia spontaneously resolved 3 weeks after the initiation of CAB therapy.

and phosphorylation of the type I TGF-β receptor (TGF-β RI) to form a complex [START_REF] Massagué | TGF-beta signal transduction[END_REF]. Once activated, TGF-β RI triggers the formation of a heteromeric complex between Smad2, Smad3 and Smad4, which ultimately translocates into the nucleus to regulate expression of various transcription factors [START_REF] Heldin | TGF-β signalling from cell membrane to nucleus through SMAD proteins[END_REF]. Sarkar et al. was the first to show that TGF-β1 inhibits, in a similar manner than dopamine, both PRL secretion and proliferation of lactotrophs [START_REF] Sarkar | Transforming growth factor-beta 1 messenger RNA and protein expression in the pituitary gland: its action on prolactin secretion and lactotropic growth[END_REF]. More recently, it was shown that, knocking out the D2DR expression or using a D2DR antagonist (sulpiride) resulted in the decrease of both TGF-β1 secretion and TGF-β RII expression by the lactotroph cells. These observations could suggest that the inhibiting effects of dopamine could be due, at least partially, to the TGF-β system [START_REF] Recouvreux | Active and total transforming growth factor-β1 are differentially regulated by dopamine and estradiol in the pituitary[END_REF]. Interestingly, in a series of 12 patients who underwent surgery because of resistance to BRC (failure of PRL normalization after at least 3 months of BRC ≥ 15 mg/day), a significant down-regulation of the TGF-β/ Smad signaling cascade assessed by high-content screening techniques (qRT-PCR, western blot, immunofluorescence and ELISA) was observed as compared to normal human anterior pituitaries [START_REF] Li | The role of TGF-β/Smad signaling in dopamine agonist-resistant prolactinomas[END_REF]. However, a comparison with DA-sensitive tumors was unfortunately lacking in this work.

Because estradiol is known to increase PRL secretion and sustained lactotroph proliferation, a combined treatment with TGF-β1 and fulvestrant, a selective estrogen receptor degrader, was tested in the rat GH3 somatolactotroph cell line. Of interest, a significant cytotoxicity in a dose-and time-dependent manner was observed with a simultaneous activation of Smad3 [START_REF] Li | The role of TGF-β/Smad signaling in dopamine agonist-resistant prolactinomas[END_REF].

Overall, decreased TGF-β1 activity and decreased expression of different components of the TGF-β1 system have been described in animal models of Prolactinomas as well as in human Prolactinomas [START_REF] Recouvreux | The pituitary TGFβ1 system as a novel target for the treatment of resistant prolactinomas[END_REF]. Taking into account that TGFβ1 inhibits lactotroph proliferation and PRL synthesis and secretion, recovering local TGF-β1 activity may be one avenue worth exploring in case of DA-resistant Prolactinomas (Figure 2).

Resistance-to-DA in the setting of genetic syndromes

A condition that seems to be associated with a higher risk of resistance-to-DA is when the PRLoma developed in the setting of a multiple endocrine neoplasia type 1 (MEN1) syndrome. The latter is a genetic disease that predisposes carriers to development of various endocrine tumors including PiTNETs in 30-50% of patients [START_REF] Marini | Multiple endocrine neoplasia type 1[END_REF]. Prolactinomas are the most frequent PiTNETs phenotype seen in MEN1 with 60% of cases. Moreover, MEN1-mutated (MEN1mut) patients seem to be less responder to DA as compared to their non-mutated counterparts [START_REF] Salenave | Macroprolactinomas in children and adolescents: factors associated with the response to treatment in 77 patients[END_REF][START_REF] Vergès | Pituitary disease in men type 1 (MEN1): data from the France-Belgium MEN1 multicenter study[END_REF]. We are still lacking molecular explanations to decipher why and how MEN1 mutations lead to a lower sensitivity to DA; however, it is noteworthy to mention that histologically, MEN1mut Prolactinomas are more frequently invasive as compared to non-mutated Prolactinomas [START_REF] Vergès | Pituitary disease in men type 1 (MEN1): data from the France-Belgium MEN1 multicenter study[END_REF][START_REF] Trouillas | Pituitary tumors and hyperplasia in multiple endocrine neoplasia type 1 syndrome (MEN1): A case-control study in a series of 77 patients versus 2509 non-MEN1 patients[END_REF]. Evolution of Prolactinomas in MEN1mut patients are characterized by a younger age at onset, a more aggressive behavior as reflected by a higher propensity to invade surrounding structure and, in the scope of this review, a lower to DA [START_REF] Delemer | MEN1 and pituitary adenomas[END_REF][START_REF] Subasinghe | Giant prolactinoma of young onset: a clue to diagnosis of MEN-1 syndrome[END_REF].

Besides MEN1, Prolactinomas can also be developed in the setting of a Familial Isolated Pituitary Adenomas (FIPA) syndrome where they represent 10% of all tumor phenotypes and even 25% when the Arylhydrocarbon Interacting Protein (AIP) gene is mutated [START_REF] Beckers | familial isolated pituitary adenomas (FIPA) and the pituitary adenoma predisposition due to mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene[END_REF]. In AIP-mutated Prolactinomas, 77% of patients were male with larger tumors, less sensitive to DA as compared to non-mutated patients [START_REF] Beckers | familial isolated pituitary adenomas (FIPA) and the pituitary adenoma predisposition due to mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene[END_REF].

Other molecular pathways involved in DA-resistance

Several other molecular pathways have been incriminated in the occurrence of DA-resistance in Prolactinomas. For instance, the expression of Filamin-A, a cytoskeleton protein with scaffolding properties, is downregulated in parallel to the one of D2DR in human DA-resistant Prolactinomas [START_REF] Peverelli | Filamin-A is essential for dopamine D2 receptor expression and signaling in tumorous lactotrophs[END_REF]. Moreover, silencing the Filamin-A expression in human DA-sensitive Prolactinomas resulted in a significant decrease of D2DR membranous expression and abrogation of the DA-induced inhibition of PRL release and antiproliferative signals. In a reciprocal way, a restoration of the D2DR expression and PRL responsiveness to DA occurred when Filamin-A was overexpressed in DA-resistant Prolactinomas [START_REF] Peverelli | Filamin-A is essential for dopamine D2 receptor expression and signaling in tumorous lactotrophs[END_REF]. Besides Filamin-A, a recent study showed that low levels of PRB3 mRNA were observed in case of DAresistant Prolactinomas and exposed the patients to a higher risk of tumor recurrence [START_REF] Wang | Low levels of PRB3 mRNA are associated with dopamine-agonist resistance and tumor recurrence in prolactinomas[END_REF]. However, the exact role of the PRB3 protein in Prolactinomas remains, to date, elusive.

Therapeutical management of resistant-to-DA prolactinomas

In the case of resistant-to-DA Prolactinomas, several options can be discussed; all of them being either proposed alone, combined and/or in a sequential approach. It includes switch to another DA, surgery, external radiotherapy, other-than-DA medical therapies or a watch-and-wait attitude.

Switching to another dopamine agonist

Since its commercialization, CAB is usually preferred to BRC for the treatment of patients with Prolactinomas because of overall greater efficacy and tolerability. However, in certain regions of the world, BRC is still used as a first choice because of costs and/ or stock availabilities. Over the past years, CAB showed a greater efficacy as compared to BRC for the control of PRL oversecretion and has been successfully tested in patients considered as being resistant to BRC [START_REF] Webster | A comparison of cabergoline and bromocriptine in the treatment of hyperprolactinemic amenorrhea. cabergoline comparative study group[END_REF][START_REF] Huang | Cabergoline for the treatment of bromocriptine-resistant invasive giant prolactinomas[END_REF][START_REF] Santos Nunes | Cabergoline versus bromocriptine in the treatment of hyperprolactinemia: a systematic review of randomized controlled trials and meta-analysis[END_REF][START_REF] Delgrange | Effects of the dopamine agonist cabergoline in patients with prolactinoma intolerant or resistant to bromocriptine[END_REF]. Low posology of CAB could, likewise, significantly overcome the resistance to QNA in Prolactinomas [START_REF] Colao | Prolactinomas resistant to standard dopamine agonists respond to chronic cabergoline treatment[END_REF], a drug which is already effective in case of BRC resistance. However, this result could be overestimated because of better compliance and tolerance to CAB as compared to QNA, which actually induces a bias in the statistical analysis. In resistant-to-CAB patient, the most common medical approach is therefore to increase the dose of CAB, as long as a reduction in PRL levels can be demonstrated with each stepwise increase [START_REF] Paepegaey | Cabergoline tapering is almost always successful in patients with macroprolactinomas[END_REF][START_REF] Gillam | The novel use of very high doses of cabergoline and a combination of testosterone and an aromatase inhibitor in the treatment of a giant prolactinoma[END_REF]. Although it is unlikely that patient resistant to CAB will demonstrate sensitivity to BRC, there is an existing clinical report of two patients resistant-to-CAB with micro-and macroprolactinoma, respectively, who unexpectedly showed response to BRC [START_REF] Iyer | positive prolactin response to bromocriptine in 2 patients with cabergoline-resistant prolactinomas[END_REF].

Surgical treatment of prolactinomas

Before the development of DA, surgery was historically the treatment of choice for the cure of a PRLoma [START_REF] Gillam | Advances in the treatment of prolactinomas[END_REF]. Currently, indications are mainly represented by cases of pituitary apoplexy, resistanceto-DA or by personal choice of the patient [START_REF] Kreutzer | Operative treatment of prolactinomas: indications and results in a current consecutive series of 212 patients[END_REF]. Even in cases of visual defect due to a macroprolactinoma, the standard of care favors the use of DAs as compared to surgery. The visual defect generally improves in the short term under DAs in a majority of patients, including those with giant (i.e. maximal diameter ≥ 4 cm) Prolactinomas and in spite of a partial antitumoral effect [START_REF] Delgrange | Giant prolactinomas in women[END_REF][START_REF] Shimon | Giant prolactinomas larger than 60 mm in size: a cohort of massive and aggressive prolactin-secreting pituitary adenomas[END_REF][START_REF] Shimon | Effectiveness of long-term cabergoline treatment for giant prolactinoma: study of 12 men[END_REF][START_REF] Corsello | Giant prolactinomas in men: efficacy of cabergoline treatment[END_REF]. However, it should be noted that there is no dedicated studies which compared the outcome of the visual field defect in patients with macroprolactinoma when treated by DA as compared to patient treated in first-line by transphenoidal surgery. In highly trained hands, selective adenomectomy results in normalization of PRL levels in 75-90% of microprolactinomas. The multi-invasive nature of macroprolactinomas obviously affects the rate of cure obtained by purely surgical in such tumors which is achieved in around 40% of cases with recurrence rates of 20% over 10 years [START_REF] Hamilton | Surgical outcomes in hyporesponsive prolactinomas: analysis of patients with resistance or intolerance to dopamine agonists[END_REF][START_REF] Dallapiazza | Outcomes of endoscopic transsphenoidal pituitary surgery[END_REF][START_REF] Jan | Prolactinoma surgery[END_REF]. Surgery will provide the tumor histological characteristics, especially its pathological markers of aggressivity (Ki67 and P53 immunostaining, mitotic count). A recent retrospective study analyzed the surgical outcome of 184 men with surgically treated Prolactinomas. Among them, 178 patients had a macroprolactinoma (152 had a maximal size below 4 cm and 26 with a maximal size >4 cm) and 61/152 (33.1%) of cases were invasive [START_REF] Song | Surgical treatment for male prolactinoma[END_REF]. A post-surgical initial remission of PRL levels was observed in only 10% of invasive cases (i.e. suprasellar and/or parasellar and/or sphenoidal invasion) [START_REF] Song | Surgical treatment for male prolactinoma[END_REF]. In case of resistance to DA, surgery is a valuable therapeutic option which results in normalization of PRL in half of patients [START_REF] Primeau | Outcomes of transsphenoidal surgery in prolactinomas: improvement of hormonal control in dopamine agonist-resistant patients[END_REF]. In expert pituitary center, surgery for macroprolactinomas can be complicated by a diabetes insipidus in around 5% of cases, cerebro-spinal-fluid leak in 2-10% and anterior pituitary deficiencies in 1-15%, respectively [START_REF] Dallapiazza | Outcomes of endoscopic transsphenoidal pituitary surgery[END_REF][START_REF] Donegan | Surgical outcomes of prolactinomas in recent era: results of a heterogenous group[END_REF].

External radiotherapy

External radiotherapy is usually discusses in patients who have clinically significant and symptomatic elevation of serum PRL in spite of DA therapy and/or transsphenoidal surgery (which means a third-line therapy in majority of cases). It can be proposed either as external beam radiation therapy (conformal radiotherapy) or stereotactic radiosurgery (SRS). Nowadays, the latter is preferably employed as its three dimensional approach is associated with a more rapid correction of hormone oversecretion and a lower risk of radiation-induced neoplasms and carotid stenosis [START_REF] Castinetti | Role of stereotactic radiosurgery in the management of pituitary adenomas[END_REF]. Amongst SRS, gamma knife radiosurgery (GKRS) provides a highly conformal and selective therapeutic intervention in a single procedure performed with image guidance and using a multiheaded cobalt unit or a linear particle accelerator. On the opposite, the conformal radiotherapy is characterized by several fractions over the time (with generally daily administration). In Prolactinomas, previous studies, including one from our group, reported a mean rate of PRL normalization ranging from 26 to 43% with a time to remission comprised between 24 and 96 months [START_REF] Castinetti | Long-term results of stereotactic radiosurgery in secretory pituitary adenomas[END_REF][START_REF] Pouratian | gamma knife radiosurgery for medically and surgically refractory prolactinomas[END_REF][START_REF] Ježková | Use of the Leksell gamma knife in the treatment of prolactinoma patients[END_REF]. In cases of resistance/intolerance-to-DA or invasive macroprolactinomas, a normalization of PRL level was obtained in half of 38 patients with a mean and median prescribed doses which were 21.3 and 25 Gy to the 50% isodose line, respectively. These doses are the most common used for GKRS in Prolactinomas [START_REF] Tanaka | gamma knife radiosurgery for patients with prolactin-secreting pituitary adenomas[END_REF]. Even if comparative studies with a significant number of patients are lacking, there are several lines of evidence which showed that withdrawal of DA therapy during SRS could be associated with better outcomes in terms of endocrine remission [START_REF] Pouratian | gamma knife radiosurgery for medically and surgically refractory prolactinomas[END_REF][START_REF] Cohen-Inbar | Gamma knife radiosurgery for medically and surgically refractory prolactinomas: long-term results[END_REF]. It is noteworthy to mention that cavernous sinus invasion was shown to be a significant negative prognosticator of endocrine remission [START_REF] Cohen-Inbar | Gamma knife radiosurgery for medically and surgically refractory prolactinomas: long-term results[END_REF]. Finally, after 40 months, radiotherapyinduced hypopituitarism occurred in 30% of cases.

Besides stereotactic radiotherapy, a conformal external radiotherapy can be discussed with the risk of adverse effects which is proportional to maximal dose and to fractionated dose per day. As compared to GKRS, conformal radiotherapy has two main drawbacks: the first one is the time to remission, equals to 5-10 years which is longer than that observed with the GKRS and therefore requires an effective medical treatment during this period of therapeutic latency. The second drawback of conformal radiotherapy is the risk of side effects, including hypopituitarism (in more than 80% of cases), optic neuritis, radiation-induced cerebral tumors, cerebral infraction and/or cognitive dysfunctions. These latter side effects occurred after a mean time of 10-20 years, and were not (at least not yet) described with the use of GKRS.

The antitumor efficacy (which means stabilization and/or decrease of the tumor residue) of radiotherapy in the setting of Prolactinomas is high, observed in 70 to 100% of cases over a delay ranging from 12 to 36 months [START_REF] Pouratian | gamma knife radiosurgery for medically and surgically refractory prolactinomas[END_REF][START_REF] Castinetti | Gamma knife radiosurgery in pituitary adenomas: why, who, and how to treat?[END_REF]. This suggests that GKRS could be a valid alternative treatment in cases of intolerance or resistance-to-DA. Of note, median time to remission was usually 20 to 40 months, which means that gonadal steroid hormones administration can be necessary during this period [START_REF] Regis | Radiosurgery: a useful first-line treatment of prolactinomas?[END_REF].

Medical treatment of DA-resistant prolactinomas

Temozolomide

In the setting of Prolactinomas, temozolomide (TMZ) never represents a first line treatment, however its indication can be actively discussed when the tumor is not controlled by the usual therapeutic regimen (DA, surgery, radiotherapy). Its efficacy was first demonstrated in glioblastomas before its successful use in advanced melanomas and neuroendocrine tumors [START_REF] Stupp | Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma[END_REF]. TMZ is an alkylating chemotherapy, derivative of dacarbazine, with lipophilic properties allowing it to cross the blood-brain barrier and acts by inserting a methyl group to DNA bases (mainly guanine). By this way, it inhibits the gene transcription and cellular replication [START_REF] Zhang | Temozolomide: mechanisms of action, repair and resistance[END_REF]. O(6)-methylguanine methyltransferase (MGMT) is an endogenous DNA repair enzyme that can remove this methyl group and thereby potentially counteracts the cytotoxic effect of TMZ. An inverse correlation has been shown between the degree of MGMT expression, due to silencing of the MGMT gene by methylation of its promoter, and the response to TMZ treatment in glioblastomas [START_REF] Hegi | MGMT gene silencing and benefit from temozolomide in glioblastoma[END_REF]. With 100% oral bioavailability, the standard regimen for administering TMZ consists in an oral daily dose of 150-200 mg/m2 body surface area for 5 days every 28 days. TMZ was proposed as a salvage therapy for the first time in 2006 to treat a PRL-secreting pituitary carcinoma [START_REF] Lim | Temozolomide: a novel treatment for pituitary carcinoma[END_REF] and subsequently tested in aggressive Prolactinomas [START_REF] Raverot | Temozolomide treatment in aggressive pituitary tumors and pituitary carcinomas: A French multicenter experience[END_REF]. In the latter and in PRLsecreting carcinomas, an overall control of tumor growth and PRL secretion under TMZ was observed in 66 and 73% of cases, respectively [100]. Moreover, patients carrying aggressive Prolactinomas who were responders to TMZ showed to have an improved overall survival as compared to their non-responders counterparts [101]. In our own experience, we previously reported a remarkable efficacy of the TMZ regimen in a patient with a MEN-1 pituitary carcinoma (Figure 3 In summary, TMZ is currently a suitable therapy that can be used as a first-line chemotherapy in case of aggressive and DA-resistant macroprolactinomas or pituitary carcinoma with tumor growth [103].

Alternative medical therapies

Other medical therapies can be discussed in a personalized approach when the PRL-secreting PiTNET displays signs of resistance to DAs. Somastostatinergic analogs (octreotide and lanreotide) which bind the somatostatin receptor subtype 2 (SSTR2) are generally inefficient to inhibit PRL secretion and/ or decrease the tumor volume [104]. Nonetheless, there are few clinical reports of DA-resistant Prolactinomas which showed that combination of octreotide plus CAB could further inhibit both PRL secretion and tumor volume [105,106]. We previously hypothesized that a chimeric compound made of SSTR2 and D2DR moieties, called dopastatin, could further inhibit PRL secretion in SST2-overexpressing DA-resistant Prolactinomas. Unfortunately, the inhibitory effect observed with dopastatin was similar to the one obtained with CAB [107]. Pasireotide (SOM230) is another somatostatin analog with high affinity for SSTR1, 2, 3 and 5. It is currently proposed in clinic for the treatment of acromegaly and Cushing's disease [START_REF] Cuny | Medical therapies in pituitary adenomas: current rationale for the use and future perspectives[END_REF]108]. In vitro, SOM230 inhibits PRL secretion from primary culture of human PRL-secreting PiTNETs [109,110] and was successfully tested in a patient with DA-resistant PRLoma [111]. In the latter, the tumor expressed high level of SSTR5, a condition which remains exceptional in DA-resistant Prolactinomas and explains why SOM230 is most of the time ineffective in such cases [104]. Finally, peptide receptor radionuclide therapy (PRRT) with 111In-DTPA-octreotide has shown interesting result in a man with uncontrolled giant PRLoma resistant to conventional therapy [112] and could, in the era of the recent provision of 177Lu-DOTATATE (LUTHATERA®) [113], represent an interesting treatment to discuss in specialized center.

Two recent cases of patients with BRC-resistant Prolactinomas suggest that metformine, an oral treatment commonly used in type 2 diabetes, can lead to normalization of PRL level when it is used in combination with BRC, and also to a subsequent decrease of the tumor volume after 24 months of treatment [114]. These results are supported in vitro by a synergistic inhibitory effect of metformine and BRC in combination over tumor growth and PRL secretion of xenograft models of lactotroph tumors [115].

4.5. New therapeutical perspectives for DA-resistant prolactinomas 4.5.1. The EGF/EGFR system as a potential therapeutic target

The epidermal growth factor (EGF) system comprises several transmembrane tyrosine kinase receptors known as EGFR (ErbB1, HER1), ErbB2 (HER2), ErbB3 (HER3) and ErbB4 (HER4) which, once binded by EGF, form either homo-and/or heterodimers, and activation of the intrinsic kinase domain and intracellular signaling [116]. EGFR is expressed in human Prolactinomas even though the pattern of EGFR expression in Prolactinomas seems to be heterogeneous and unconstant from one tumor to another [117][118][119]. In experimental models, gefitinib, an EGFR antagonist, decreased i/the cell proliferation of the rat somatolactotrope GH3 cell line ii/the PRL mRNA expression, iii/the PRL-secreting xenograft volume in rodents and the PRL secretion in vivo [120]. In humans, two patients with DA-resistant Prolactinomas were treated with lapatinib (1,250 mg daily for 6 months), a tyrosine kinase inhibitor of ErbB-1 and -2. One of them showed a significant improvement of his PRL level (from 311 to 67 ng/ml) together with a regression of the central portion of the tumor mass, albeit incomplete. In the second patient, lapatinib was moderately effective with a decrease of PRL level from 447 to 259 ng/mL (at the last follow-up) however without any shrinkage of the tumor mass [118].

The RAF/MEK/ERK and PI3K/AKT/mTOR signaling pathways in prolactinomas

The RAF/MEK/ERK and PI3K/AKT/mTOR signaling pathways are crucial to many aspects of cell growth and survival, in physiological as well as in pathological conditions [121]. Previously, no differences were found between PA and normal pituitary samples in the expression of phosphorylated/total mTOR, TSC2 or p70S6K known to be the downstream effectors of the PI3K/AKT signaling pathway [122]. As such, the PI3K/AKT/ mTOR signaling pathway does not seem to represent a canonical pathway involved in the pathogenesis of PA, especially Prolactinomas. On the opposite, the expression of the phosphorylated forms of MEK1/2 and ERK1/2, the downstream effectors of the MAPK signaling pathway, was significantly higher in Prolactinomas as compared to normal pituitaries [122]. Interestingly, a recent study suggests an intricate cross-talk between the ERK and PI3K signaling pathways in a rat somatolactotrope cell line GH4T2, in which PI3K appears as a counterregulatory mediator of the ERK-induced PRL transcription [123]. In the same cell line, the inhibition effect obtained with CAB over PRL secretion and cell proliferation was mediated through the phosphorylation of S6K, a target of mTOR which therefore seems to be closely involved in the response to DA in Prolactinomas. Besides the response to DA, the mTOR signaling pathway was recently identified as a promotor of pituitary tumor development, more specifically in Prolactinomas [124] and in vitro data suggest that rapamycin, a mTOR inhibitor, could be effective in the treatment of lactotroph tumoral cells [125]. Recently, a patient with DAresistant PRLoma was successfully with everolimus, a mTOR inhibitor, with a significant decrease of both PRL levels and tumor volume after 5 months [126]. The tumor immunohistochemical analysis of this patient revealed high level of p-AKT, p4EBP1 and p70S6K [126]. In conclusion, pharmacological inhibitors of the ERK or PI3K pathways could constitute an interesting therapeutic approach in DA-resistant Prolactinomas.

Summarizing conclusions

In conclusion, Prolactinomas represent the most common hormone-secreting pituitary tumor with an overrepresentation of microadenomas in the population of young women. Difficult-to-treat Prolactinomas are represented by

Prolactinomas that are resistant to DA therapy a condition which is correlated to the occurrence of a macroadenoma in more than 80% of cases [START_REF] Vroonen | Prolactinomas resistant to standard doses of cabergoline: a multicenter study of 92 patients[END_REF]. Because these tumors can be roughly assimilated to aggressive tumors, a multidisciplinary team meeting is obviously required to guide the best therapeutical approach to propose [103].

The above manuscript gives an overview of the valuable treatments to discuss when the patient does not respond (enough) to DA. In clinical practice, two situations can be distinguished. First presentation (which is the more frequent): the patient keeps a significant oversecretion of PRL after surgery, as it occurs in 60% of macroadenomas [START_REF] Primeau | Outcomes of transsphenoidal surgery in prolactinomas: improvement of hormonal control in dopamine agonist-resistant patients[END_REF]127] but the tumor does not display features of aggressivity on the pathological analysis and does not exert a tumor mass effect on surrounding structures. The concern is mainly related to the effect of hyperprolactinemia, especially hypogonadism. A step-by-step therapeutic approach is generally adopted with increasing the dose of CAB, and potentially a subsequent radiotherapy. In the meanwhile, an hormonal substitution can be necessary with periodical follow up by pituitary It is important to underline that the risk of cardiac valvulopathy while increasing the dose of CAB is rare. In our daily practice, we recommend, as others do, to perform one echocardiography at baseline, which, if normal, will require a subsequent control in the future if the patient has an audible murmur, if he is treated for more than 5 years at a dose of more than 3 mg per week, or for those who maintain CAB treatment after the age of 50 years [128]. When the tumor is aggressive according to Trouillas criteria [129], the concern is rather related to its (uncontrolled) invasive behavior and requires urgent therapeutic management. Repeated surgery, MGMT immunostaining and ultimately TMZ can be proposed with a close monitoring. Once PRL is normalized, and as long as it remains normal or stable overtime, the likelihood of a significant tumor growth remains low, and we therefore assume that repeated pituitary MRI in the follow up are unnecessary [130].

In summary, different therapeutic modalities are discussed during the care of a patient with a DA-resistant macroprolactinoma. Selected therapeutic perspectives have been discussed and detailed in this review, but many others are in the pipeline and could be of interest in PRL-secreting PiTNETs. This is the case for the immunotherapy whose pharmalogical action rather targets the microenvironment than the tumor niche itself. A spectacular antitumoral effect of the combination of ipilimumab (anti-CTLA4) and nivolumab (Anti-PD1), was observed in a case of ACTH-secreting pituitary carcinoma [131]. The impact of the immune components in the pathogenesis, behavior and response-to-treatment of Prolactinomas remains largely unknown. Therefore, a better understanding of the molecular and cellular factors underlying the resistance to DA in Prolactinomas could allow in the future to personnalize the treatment for a patient and optimize his medical care.

Expert opinion

Dopamine agonist (DA) treatment represents the first-line therapy for Prolactinomas either micro-or macroadenomas and as such, their overall efficacy is one of the most remarkable observed in the field of medicine. Even when a single weekly dose of CAB is unsufficient, it is worth a try to increase the posology given the fact that a certain proportion of macroprolactinomas will respond at higher than conventional dose. Based on our experience, some clinical indicators are, from the moment of the diagnosis, are already highly predictive of resistance to DA: a (really) young age (i.e. <18 years old) at diagnosis, the existence of a MEN1 or less frequently AIP germline mutation, and a giant adenoma (which means maximal diameter exceeding 4 cm). In the latter situation, a tumor mass effect usually occurs on the optic chiasm, likely responsible of visual field defect. The authors want to underline that CAB therapy, even when a state of resistance is observed with no reduction of the tumor volume, a dramatic improvement of the visual field can be observed, suggesting that the treatment with CAB can modify the consistency of the tumor making it more soft. This hypothesis is also supported by the possible occurrence of intratumoral hemorrhage during the treatment with CAB.

We usually recommend a careful assessment of the DA efficacy on both PRL secretion and tumor volume reduction by MRI, 4 months after initiation of the therapy and then 6 to 8 months later.

As such, we recommend to wait for 12 months of treatment under CAB before considering the tumor is resistant to DA.

The different works that have been published so far to decipher the molecular abnormalities involved in the resistance to DA in Prolactinomas have sparked many interest for the pharmacological research however without concrete clinical applications yet. Indeed, in the case of treating DA-resistant tumor (debulking) surgery is the prior therapeutic option more or less followed by stereotactic radiotherapy on the tumor residue. This therapeutical strategy at least offers to the clinician the access to the tumor histopathology and especially to its aggressive behavior. This last point is important in case of multiple relapse and uncontrolled growth of the tumor which therefore could indicate treatment with TMZ. The results obtained on tumoral residue with gamma knife radiosurgery (GKRS) are encouraging in a majority of patients when performed in an expert center, with few side effects and a prolonged control of the tumor volume and hormonal secretion. The current alternative therapies to dopamine agonist are almost nonexistent and only few ones have shown an interesting effect in case reports. This is the case with somatostatin analogs including pasireotide, whose use could be attempted when the tumor expressed high levels of somatostatin receptor subtype 5. Similarly, the use of Lapatinib could be of interest but its efficacy has been demonstrated in very few cases of resistant Prolactinomas. Overall therapies directed toward membrane receptors do not appear to us as the preferential targets to develop in case of DA-resistant or only in a cotargeting therapeutical approach. On the opposite, promising approach are represented by inhibitors of canonical signaling pathways situated downstream the transmembrane receptors (e.g. MAPK) as well as the development of epigenetic drugs that could lead to the re-expression of D2DR when this one is down-regulated.

A subgroup of patient will present with a tumor residue of their DA-resistant Prolactinomas however without significant progression. As long as the tumor does not lead to tumoral and/or hormonal symptoms, a regular follow-up can be sufficient in the management of such patients. In all the remaining cases (i.e. when a significant progression is observed over the time) a multidisciplinary approach is of upmost importance to discuss which therapeutical strategy presents the best risk/ benefit balance for the patient in regards to his/her clinical profile.

We, and others, assume that the therapeutical management of difficult to-treat macroprolactinomas will benefit, instead of increasing the posology of DA, from identification of new therapeutical targets in case of DA-resistance. As such, the recent works conducted by the group of Melmed investigated drugs directed against the Erb receptor as a reliable treatment to consider. Likewise, it is worth trying to assess the efficacy of pasireotide in tumors expressing significant level of somatostatin receptor subtype 5. Finally the coming years will probably also marked by new stereotactic radiosurgery procedures that will undoubtedly optimize the treatment of PRLoma residues.

Figure 2 .

 2 Figure 2. Schematic illustration of the main signaling pathways involved in the response to dopamine agonist therapy in pituitary lactotrophs. In the sake of clarity, major signaling pathways are simplified and readers can refer to the review for more details. On the right side of the cell, optional therapies and the ones that are in development are represented. The question mark means a possible effect supported by in vitro experiments.

Figure 3 .

 3 Figure 3. Coronal T1-weighted pituitary MRI in patient with a DA-resistant prolactinoma treated with temozolomide. [A] In 1995, after pituitary surgery, showing postoperative sequellae without any large visible residual adenomatous tissue. [B] In 2006, showing a relapse of the tumor with a large adenomatous residue in the left cavernous sinus. [C] In 2011, after 24 sessions of temozolomide (given between 2006 and 2008) with prolactin decreasing from 6950 to 98 ng/mL and the tumor volume from 62%, at the last follow-up.

Table 1 .

 1 Overview of the main studies summarizing the efficacy of dopamine agonist in patients with macroprolactinoma and/or primary resistance to bromocriptine. BRC, bromocriptine; QNA, quinagolide.

		Major side effects Ref.			None [132]			4.7% stopped the treatment [133]				None [80]				None [28]				None [46]	3/18 CSF leak [78]			One case of CSF leak after initiation of [134]	therapy that resolved upon dose	reduction.	CSF leak occured in 8 patients [35]				None [135]		4 CSF leak (11,76%) [31]	One CSF leak who underwent surgery [136]	2 patients present severe psychiatric [41]	disorders (severe depression and one	suicidal ideas	(Continued )
	Tumor volume decrease (TVD) and	disappearance (TD)			TVD ≥ 80% in 61% patients at	24 months.	TD in 6 patients at 12 months	TVD ≥ 25% in 53% patients at	15 months	TD in 12.9% patients at	15 months	TVD ≥ 25% in 90% patients at	12 months			TVD: 100% at 24 months (reducing	in diameter up to 73.7%)	TD: 30% at 24 months.		NR	TVD >90% in 8 patients	33% < -90% 7/18 reduction	>33% at 7,8 years	TVD >50% in 87% patients	TD: in 11% of patients		NR				NR		TVD: 79% under CAB >30%	reduction	TVD: 100% at 135 months (mean	98% decrease)	TD: 76% had almost completely	disappeared tumor.	TVD: 75% a 31.6 months
	Inhibition of prolactin secretion in % of patients	(N: normalization; D: decrease)			N: 82% at 12-24 months.			N: 61.2% at 15 months	D ≥ 75% in 28% at 15 months			N: 50% at 12 months				N: 75.6% at 24 months.	10 patients classified as resistant (non-normalization with	CAB>2 mg per week), dose increased to 7 mg per week without	significant decrease.	N: 98.2% at 12 months	N: 11/18 patients (median: 20 months)	D: 6/18 patients		N: 68% at 3,5 years	D: 19% decreased <100 ng/mL		N: 60.8% of the 220 patients	N: 71.6% of the 116 patients			N: 60% at 7.3 years		N: 7/15 (47%) under BRC	N: 10/18 (55%) under CAB	N: 1/1 (100%) under Pergolide	N: 76% patients (at 135 months).	N: 67% at 31.6 months
		Population of the study			N = 23 prolactinomas	3 operated before CAB treatment	8 previously treated with BRC	N = 85 macroprolactinomas	18.8% resistant to BRC	37.6% intolerant to other DAs		N = 10 prolactinomas	4/10 operated before CAB therapy,	without modifying the prolactin levels	or the tumor size.	N = 41 macroprolactinomas	Only men			N = 57 macroprolactinomas	N = 18 giant PRL-secreting adenomas	(max size >60 mm)	NB: 1 patient treated with BRC 15 mg/day	N = 47 giant prolactinomas			N = 260 macroadenomas	220/265 were treated with CAB	116/220 had only CAB		N = 100 macroprolactinomas	65 patients have been treated by	DAsalone	N = 34 giant prolactinomas in women	Follow-up 3 to 198 months	N = 25 giant prolactinomas	N = 12 macroprolactinomas
		Dopamine agonist (DA)	CABERGOLINE (CAB)	(dose/week)	0.25 to 2 mg			0.25 to 10.5 mg				1.5 to 10.5 mg				0.5 to 7 mg				0.5 to 12 mg	1 to 8 mg			1 to 5 mg			0.5 to 8 mg		OTHER DOPAMINE	AGONISTS	BRC (dose 17.5 to	105 mg/week)	CAB (dose 0.25 to 6/	week)	BRC (15/34 patients,	dose: 5 to 30 mg/	day)	CAB (18/34 patients,	dose from 0.5 to	7 mg/week)	One patient with	pergolide	BRC (24/25 patients,	dose from 2.5 to	15 mg per day)	CAB (in 1/25 patient,	dose 2 mg/week)	QNA (dose from 0.075	to 0.4 mg per day)

Table 1 .

 1 (Continued).

		Ref.	[137]
		Major side effects	11 (10.2%) patients had to discontinue	treatment because of adverse	reactions.
	Tumor volume decrease (TVD) and	disappearance (TD)	TVD: 100% in all patients (mean	31%)	TVD >50% in 19.5% of patients	TVD: 20/58 patients in the	subgroup of macroadenomas	without radiotherapy	TVD >50%: 11/20 patients	At 6 months	TVD >80% in 4/16 patients with	TD in 2 patients in the QNA	group	TVD >80% in 5/14 patients with	TD in 2 patients in the CAB group
	Inhibition of prolactin secretion in % of patients	(N: normalization; D: decrease)	N: 44% at 9.8 months
		Population of the study	N = 107 patients previously treated with	BRC and considered as BRC-resistant.	80 patients had a macroadenoma					N = 16 macroprolactinomas	12 months QNA next to 12 months CAB	with a washout period.	Note all patient reincreased their PRL	levels in the wash-out period.
		Dopamine agonist (DA)	QNA (dose from 0.075	to 0.75 mg per day)						QNA (dose from 0.075	to 0.6 mg/day)	CAB (dose from 0.5	to 3 mg/week)

N: 14/16 for QNA At 12 months N: 14/16 for CAB at 12 months


Teddy Fauquier

Nathalie C Guérineau

R Anne Mckinney

Karl Bauer

Patrice Mollard
email: mollard@u469.montp.inserm.fr

A new route for long-distance communication in the anterior pituitary

Keywords: folliculostellate cells, [Ca 2+ ] i , cytosolic calcium concentration, TTX, tetrodotoxin, TEA, tetraethylammonium, 4-AP, 4-aminopyridine, PPADS, pyridoxalphosphate-

All higher life forms critically depend on hormones being rhythmically released by the anterior pituitary. The proper functioning of this master gland is dynamically controlled by a complex set of regulatory mechanisms that ultimately determine the fine tuning of the excitable endocrine cells, all of them heterogeneously distributed throughout the gland. Here we provide evidence for a new intrapituitary communication system by which information is transferred via the network of non-endocrine folliculostellate (FS) cells. Local electrical stimulation of FS cells in acute pituitary slices triggered cytosolic calcium waves, which propagated to other FS cells by signaling through gap junctions. Calcium wave initiation was due to the membrane excitability of FS cells, hitherto classified as silent cells. FS cell coupling could relay information between opposite regions of the gland. Since FS cells respond to central and peripheral stimuli and dialogue with endocrine cells, this new form of large-scale intrapituitary communication may provide an efficient mechanism that orchestrates anterior pituitary functioning in response to physiological needs.

Classically, the anterior pituitary is considered as a secondary oscillator obeying mainly hypothalamic factors, either increasing or decreasing secretion of pituitary hormones, which are released in an episodic manner at the basis of the medium eminence (1)(2)(3)(4). The portal blood vessels form the interorgan communication system driving the hypothalamic inputs to the anterior pituitary gland. They collect the transmitters released by hypothalamic nerve endings at the primary capillary plexus level and slowly distribute them in the endocrine parenchyma via the complex arborescence of pituitary sinusoids between the columnar units of pituitary cells (so-called cell cords) (5,6). Over the last three decades, many studies carried out in isolated endocrine cells have provided strong evidence that endocrine cells generate action potential-driven rises in cytosolic calcium concentration ([Ca 2+ ] i ) that are probably the keystones of dynamic adjustment of numerous cellular functions including exocytosis and gene expression (7)(8)(9). Recently, the technique of acute slice preparations applied to the anterior pituitary revealed that endocrine cells do fire short-term [Ca 2+ ] i transients due to their electrical activity in situ (10,11). Temporal organization of these transients provide a very fine mode of timing information (time-locked rhythmic bursting pattern) when a bolus of secretagogues invades the glandular parenchyma (11).

However, the activity of the gland as a whole does not reflect the average of independent dynamics of cellular messages that occur in the distinct endocrine cell types scattered throughout the tissue. In this respect, one puzzling finding is the persistence of pulsatile releasing profiles of hormones when the gland is disconnected from the hypothalamic inputs (12,13). This indicates that a large-scale communication system exists within the anterior pituitary. In spite of the wealth of information on cell-to-cell mechanisms between endocrine cells that comprise the release of paracrine factors (14) and gap junction signaling (10), spreading of spatial information that crosses the limits of single pituitary cell cords could not be explained by these mechanisms.

Since a highly efficient process spreading spatial information to the entire gland and even to pituitary sub-regions has not been reported yet, we explored here whether nonendocrine folliculostellate (FS) cells can support long-distance information transfer within the gland. FS cells display a star-shaped cytoplasmic configuration intermingled between hormone-secreting cells. The organization of FS cells within the parenchyma forms a threedimensional anatomical network, in the meshes of which the endocrine cells reside (15,16).

Very little is known about the functioning of this FS cell network, in particular with regard to the dynamics of cellular/intercellular messages. To study the behavior of this network, we measured multicellular changes in [Ca 2+ ] i , a messenger involved in a wide range of cell-tocell communication mechanisms (17)(18)(19)(20)(21)(22)(23), electrophysiological properties of FS cells and intercellular diffusion of dyes in acute pituitary slices. We showed here that the FS cell network forms a functional intrapituitary circuitry in which informationCa 2+ signals and small diffusible moleculescan be transferred over long distances (mm range) within the intact pituitary tissue.

METHODS

Cytosolic calcium monitoring in acute slices

200-µm-thick pituitary slices from 10-12-week-old female Wistar rats were prepared as previously described (11). Ringer's saline contained (in mM): NaCl, 125; KCl, 2.5; CaCl 2 , 2; MgCl 2 , 1; NaH 2 PO 4 , 1.25; NaHCO 3 , 26; Glucose, 12. Slices were incubated with 40 µM β-Ala-Lys-Nε-AMCA, a UV light-excitable fluorescent dipeptide taken up by FS cells (24), at 37°C for 1.5 hours and then exposed to 15 µM Oregon Green 488 BAPTA-1/AM (Molecular Probes) periodically delivered (3 s per 20 s for 15-20 min) onto a cell field via a blunt micropipette (11). Time-lapse optical sequences were captured with a real-time confocal microscope (Noran Odyssey XL, 488 nm-excitation wavelength, 120 images/s with averaging 4 frames), which also delivered a TTL signal that synchronized image acquisition with a brief voltage pulse (1 ms, 0.5-10 V) applied to a patch-pipette filled with Ringer's saline and positioned on the cell (10). Since the confocal microscope was fitted with an Argon/Krypton laser (visible light wavelengths), cells at the slice surface were first viewed with a Xenon lamp via the epifluorescent port of the upright microscope. Only cells that showed both β-Ala-Lys-Nε-AMCA and Oregon Green 488 BAPTA-1 were taken for subsequent [Ca 2+ ] i monitoring. In some experiments, electrical field stimulation was used to locally stimulate a cell field. A current step was shortly applied between the tips (50-100 µm apart) of two pipettes (filled with Ringer's saline) touching the slice surface. [Ca 2+ ] i changes were imaged with an intensified cooled CCD camera (PentaMAX Gen IV; Princeton Inst., Trenton, NJ) and acquired with Metafluor (Universal Imaging Corp., West Chester, PA).

Image analysis was carried out with InterVision 1.5.1, NIH Image 68k 1.58, Igor Pro 3.14, and Adobe Photoshop 5.0. Pharmacological agents were either superfused or locally applied with a puff pipette.

Patch-clamp measurements

Whole-cell electrical events were recorded using an EPC-9 patch-clamp amplifier (HEKA Electronik, Lambrecht-Pfalz, Germany). Intra-pipette solution contained (in mM): Kgluconate, 140; KCl, 10; MgCl 2 , 2; EGTA, 1.1; HEPES, 5; pH, 7.2 (KOH). Current steps were injected through the pipette tip at the soma level only. When membrane potential measurements were combined with [Ca 2+ ] i imaging, fluo-3 Ksalt (100 µM) was included and EGTA was lowered to 0.2 mM. Lucifer yellow (1 mM, Sigma) or Neurobiotin TM (N-(2aminoethyl)-biotinamide hydrochloride, 1%, Vector) was added to the intrapipette solution when appropriate. When inward currents were recorded in voltage-clamp conditions, CsCl replaced Kgluconate in equimolar amounts and BAPTA (10 mM) was used as a Ca 2+ chelator. Only cells with a high input resistance (≥ 1 GΩ), which provided acceptable spaceclamp conditions, were recorded with the voltage-clamp mode.

Immunostaining

Laminin staining was performed with a polyclonal antibody (Sigma) when the FS cell network had been traced with Neurobiotin TM (revealed with FITC-labeled avidin D).

Samples were examined with a Zeiss LSM 410 confocal microscope. Neurobiotin TM was visualized with an argon ion laser at 488 nm and a HeNe laser, pre-tuned to 543 nm, was used to image laminin simultaneously. Optical slices of 0.2 µm intervals with each image averaged were used to improve the signal-to-noise ratio. Dual staining was reconstructed three-dimensionally using Imaris Software (Bitplane AG, Zurich, Switzerland).

Vaseline-gap technique

Coronal slices were placed in a two-compartment chamber so that both slice sides bathed in separate compartments. Insulation between compartments was secured by a thin Vaseline bridge surrounding a slice portion of about 0.5-mm-width (Fig. 6b). One compartment was usually filled with β-Ala-Lys-Nε-AMCA-containing Ringer's saline, whereas the other contained peptide-free medium. Incubation for 3 hours at 37°C was carried out to achieve intense AMCA fluorescence due to peptide uptake by FS cells. Lectins labeled with fluorescent dyes were routinely included in the bathing solutions to check the watertightness of the Vaseline bridge and to help delineate the bridge borders after slice fixation.

RESULTS

Generation of calcium waves within the FS cell network. Experiments were carried out in acute rat pituitary slices (10) in order to preserve the framework of the FS cell network. We visualized FS cells by incubating slices with β-Ala-Lys-Nε-AMCA, a fluorescent dipeptide derivative that specifically accumulates in FS cells (24) (Fig. 1a). In addition, short-term (≤ 15-20 min) exposure with Oregon Green 488 BAPTA-1/AM (11) allowed Ca 2+ dye-loading of β-Ala-Lys-Nε-AMCA-positive FS cells before loading the neighboring endocrine cells to a detectable threshold (Fig. 1b). Voltage from a micropipette touching an FS cell was used to deliver local electrical stimulation (10), while multicellular changes in [Ca 2+ ] i were monitored with real-time confocal microscopy. Brief stimulation triggered a fast-peaking [Ca 2+ ] i rise (time to peak < 1 s), which was conveyed from one FS cell to the next (Fig. 1c) (156 out of 176 trials, 59 different slices). The apparent velocities of [Ca 2+ ] i waves (measured from the delays between onsets of [Ca 2+ ] i rises in FS cell somas located at the same optical plane) were 124 ± 13 µm/s (n = 207 cells, 23 fields). The [Ca 2+ ] i rises observed in FS cells distant from the site of stimulation obviously resulted from propagated [Ca 2+ ] i waves. It is highly unlikely that these [Ca 2+ ] i rises were due to direct electrical excitation since they did not occur simultaneously, but only when a stimulation was applied through a pipette in contact with an FS cell, and they did not spread uniformly in all directions. Finally, it should be noted that the FS cell network could also work as an autonomous system since [Ca 2+ ] i waves propagating spontaneously between several FS cells at a single plane of focus were occasionally observed during long-lasting optical recordings and in the absence of any stimulation electrode (data not shown).

FS cell membrane excitability is responsible for initiating calcium waves.

Establishing [Ca 2+ ] i transients in FS cells mostly depends on membrane excitability since tetrodotoxin (TTX, 0.5 µM), a specific Na + channel antagonist, reversibly blocked the [Ca 2+ ] i transients in FS cells that propagated [Ca 2+ ] i waves (Fig. 2a, 8 out of 10 cell fields). Also, Cd 2+ , a blocker of voltage-gated Ca 2+ channel currents, completely suppressed both the initial [Ca 2+ ] i rise and propagated waves (7 out of 7 cell fields, data not shown). This result was astonishing because FS cells were previously considered to be silent cells (25). We therefore examined the electrical membrane properties of FS cells with the whole-cell patchclamp technique. FS cells showed a resting potential of -61 ± 5 mV (n = 115) and displayed a remarkably wide range of input resistance (35 MΩ to 4.2 GΩ), which occasionally varied during the time period of recordings. When FS cells had a relatively high input resistance (≥ 1 GΩ), action potentials could be triggered upon step depolarization in 37 out of 70 of these cells (Fig. 2b-d). In the remainder cells recorded under current-clamp conditions, the spatio-temporal current flow along the processes might not have been strong enough to generate ectopic action potentials (23), as most FS cells emit prolonged cell processes with distal 'end-feet' (16). TTX consistently suppressed the fast component of action potentials (Fig. 2b). Smaller spikes were however observed in some TTX-treated cells (Fig. 2d). Both As the initiation of [Ca 2+ ] i waves most probably depended on action potential firing, we characterized the voltage-gated conductances expressed in FS cells. When K + currents were blocked by Cs + dialysis through the patch-clamp pipette, a fast-activating, fastinactivating TTX-sensitive inward Na + current was measured in 27 out of 43 cells (Fig. 3a).

In 51 out of 84 cells recorded in the presence of both TTX and Ba 2+ ions, step depolarization triggered voltage-gated inward currents flowing through Ca 2+ channels that were abolished by Cd 2+ ions (Fig. 3b). It should be noted that the activation threshold of Ca 2+ currents corresponded to the threshold of TTX-resistant spikes (Fig. 2d). Besides the voltage-dependent switch-off of both Na + channel and Ca 2+ channel currents, spike repolarization probably involved K + channel currents in FS cells since Ba 2+ ions, a strong K + channel blocker, markedly prolonged action potential duration (100 ± 4 ms calculated at 50% of spike repolarization, 4 out of 9 cells, data not shown). Over 57 cells recorded in the presence of Ringer's saline, step depolarization could trigger at least two distinguishable outward currents, which both were most likely carried by K + ions (mean reversal potential: -74 ± 3 mV). 86 % of cells showed a delayed outward K + current (mean activation threshold:

-25 ± 0.3 mV) (Fig. 3c), which was suppressed by 15 mM tetraethylammonium ions (TEA, n = 12). In 70% of recorded cells, a transient outward K + current with a low threshold for voltage activation (-42 ± 0.3 mV) was observed. This transient current persisted in the presence of TEA (Fig. 3d), whereas it was reduced by more than 50% by 5 mM 4aminopyridine (4-AP, n = 6, data not shown).

Calcium wave propagation depends on gap junction signaling. We then explored the mechanism that underlay [Ca 2+ ] i wave propagation. Two sources of cell-to-cell communication not mutually exclusive could be involved. Gap junctions were observed along FS cell membranes (27,28). FS cells are also known to release various products that may act on neighboring FS cells (14,29).

We first examined whether the spreading of [Ca 2+ ] i waves required gap junction signaling. Carbenoxolone, a glycyrrhetinic acid analog, reversibly reduced by 70.5 ± 10% the number of FS cells propagating [Ca 2+ ] i waves (n = 10 fields; p<0.001, paired Student's two-tailed t-test) (Fig. 4a). The action of carbenoxolone was most likely due to its efficiency at blocking gap junctional communication (30) We then investigated the possible involvement of an extracellular route in the [Ca 2+ ] i wave propagation. Purine nucleotides have been identified as an extracellular messenger in various cells including FS cells (31) and there is evidence that they may act in concert with gap junction signaling for wave propagation in cell preparations (17)(18)(19)(20)22). Bath application of both pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) and suramin (100 µM each), antagonists known to depress purinergic receptor transmission in glial cell networks (18), failed to suppress propagated [Ca 2+ ] i waves (Fig. 4c) (n = 9 fields) while they abolished [Ca 2+ ] i responses to ATP (10 µM, 10-s application, n = 23 cells, data not shown).

FS cell network: a functional large-scale communication system. In the view that we imaged propagated [Ca 2+ ] i waves with a restricted spatial resolution (square fluorescent confocal images ≤ 5,600 µm 2 ), it was important to know whether the FS cell network could transfer information over long distances within the parenchyma. [Ca 2+ ] i imaging of FS cells combined with distant electrical field stimulation (Fig. 5a) revealed prominent, coordinated transient [Ca 2+ ] i rises that occurred far away (0.5 to 1 mm) from the stimulation site (Fig. 5b, 6 slices). [Ca 2+ ] i waves, which were first recruited upon electrical stimulation and then propagated along the FS cell network, were very likely to be at the origin of the distant [Ca 2+ ] i rises, as these only occurred when stimulation intensity exceeded a threshold level (700 µA to 5 mA). Also, propagated [Ca 2+ ] i waves were closely correlated with changes in membrane potential. [Ca 2+ ] i recordings combined with patch-clamp monitoring of FS cells several hundred microns distant from the site of stimulation showed the occurrence of transient depolarization that coincided with each propagated [Ca 2+ ] i rise (Fig. 5c, n = 5 cells). Similar depolarizing events were detected upon propagated [Ca 2+ ] i rises in FS cells that were near single electrically-stimulated FS cells (n = 4, data not shown). Propagated [Ca 2+ ] i rises were always associated with transient depolarization exceeding a threshold level of about -50 mV (Fig. 5c), notwithstanding the distance between the patched cells and the site of stimulation (range: 17-520 µm, n = 9). Inversely, such a depolarization was never observed in FS cells that failed to display propagated [Ca 2+ ] i rises (n = 6, data not shown).

A final set of results provided evidence that the FS cell network forms an extended three-dimensional mode of communication throughout the gland. Consistent with this assumption, we found that [Ca 2+ ] i waves were electrically triggered in FS cells from coronal as well as saggital slices. Also, the apparent low velocity of [Ca 2+ ] i waves (17.7 ± 0.8 µm/s, n = 45 cells, 6 fields) calculated from the distance between the pair of electrode tips and recorded FS cells (Fig. 5) suggested a complex arborization of the gap junction-coupled FS cell network within the labyrinthine cell cords. Two results strengthened this proposal. First, single-cell dialysis of the Neurobiotin TM tracer traced a meshwork of connected FS cells over territories, which crossed cell cord boundaries (Fig. 6a, n = 16 fields). Second, multiple site introduction of low-molecular weight fluorescent dyes into FS cells provided further evidence for multidirectional, long-distance (mm range) transfer of information throughout the parenchyma (Fig. 6b). Bath application of β-Ala-Lys-Nε-AMCA on one side of the coronal slices (insulated from the other side with a Vaseline bridge) allowed many FS cells to take up the fluorescent dipeptide. β-Ala-Lys-Nε-AMCA diffused across the Vaseline bridge up to the opposite side of the slices (n = 9). Carbenoxolone (100 µM) blocked the spreading of β-Ala-Lys-Nε-AMCA (n = 5) which, given its small molecular weight (432.5) (24), probably diffused between FS cells coupled by gap junctions.

DISCUSSION

In this study, we provide strong evidence that the anterior pituitary possesses an intrinsic system of communication that allows long-distance transfer of information within its parenchyma. The hitherto enigmatic agranular FS cells form the framework of this communication system. These FS cells express an extended repertoire of signaling mechanisms to generate and transmit messages (regenerative [Ca 2+ ] i waves, cell-to-cell diffusion of molecules) along their cell network wiring up the gland. This work reveals that the FS cells can now be given the label "excitable cell", nearly half a century after they were discovered (15). Only one study so far has shown in cultured FS cells (TtT/GF cell line) the presence of a delayed rectifier K + channel current, which was detectable when the cloned FS cells were in contact (25). We demonstrated here that, in situ, FS cells had a set of voltage-gated conductances sufficient to generate action potentials and ensuing transient rises in [Ca 2+ ] i . To do so, FS cells displayed TTX-sensitive Na + currents and Cd 2+ -sensitive Ca 2+ currents, which both could provide a depolarization at the foot of action potential that increases the probability of spike firing. Whereas a built-in inactivation process led Na + channels to close quickly, the fast-activating, slowly-inactivating Ca 2+ channels apparently remained open long enough during single action potentials to cause a significant [Ca 2+ ] i increase. Also, two K + channel currents, which shared common properties with the 4-AP-sensitive I A and TEA-sensitive delayed rectifier K + currents [START_REF] Hille | Ionic channels of excitable membranes[END_REF], respectively, helped shape the repolarizing process of action potentials.

Excitability, which may at first appear as an atypical behavior for such agranular cells, is most likely one of the means to trigger long-range [Ca 2+ ] i signaling, a hitherto unknown mode of communication at the anterior pituitary level. Brief electrical stimulation of one or a few FS cells promoted [Ca 2+ ] i waves between FS cells over long distances across the parenchyma. Because of their sensitivity to both TTX and Cd 2+ ions, we propose that the electrical activity induced in stimulated FS cells initiated the propagation of intercellular [Ca 2+ ] i waves along the FS cell network.

A closely related question is how [Ca 2+ ] i rises traveled from one FS cell to the next.

Our results strongly suggested that, in situ, many FS cells were extensively coupled through gap junctions and that these gap junctions were responsible for the propagation of [Ca 2+ ] i rises between the coupled cells. The involvement of gap junctions in this phenomenon was fourfold. First, the gap junction blocker carbenoxolone reversibly reduced the number of FS cells propagating [Ca 2+ ] i waves that were initiated by either focal electrical stimulation or IP 3 dialysis; second, intracellular injections of Lucifer yellow traced the FS cells that propagated [Ca 2+ ] i waves; third, intercellular diffusion of small tracers (Neurobiotin TM and β-Ala-Lys-Nε-AMCA) resulted in the labeling of three-dimensional ensembles of FS cells that were also blocked by carbenoxolone; and fourth, it was highly unlikely that activation of membrane receptors, such as purinergic receptors after ATP release, be involved in [Ca 2+ ] i waves triggered following voltage stimulation.

The nature of signals flowing between gap junction-coupled FS cells that propagated electrically-induced [Ca 2+ ] i waves was then addressed. Both the velocity (124 µm/s) and extent (mm range) of [Ca 2+ ] i waves suggested that the latter spread along the three-dimensional FS cell network in a regenerative manner. Two types of regenerative signals seemed feasible: a cell-to-cell transfer of second messengers (52) or an electrical event [START_REF] Galarreta | [END_REF][START_REF] Venance | Proc. Natl. Acad. Sci. U S A[END_REF]. The first possibility appeared barely compatible with the results of this study. In FS cells, average speeds of intercellular [Ca 2+ ] i waves driven by single cell IP 3 -dialysis were slow (15 µm/s), as it has been observed for second messenger waves involving Ca 2+ release from internal stores in a chain reaction, owing to the regenerative nature of the release process (19)(20)(21)(22). Thus, second messenger waves could propagate between FS cells, but they were probably not representative of propagation mechanisms in most FS cells when an electrical stimulation was locally applied. In favor of the second possibility was the detection of membrane depolarization coincident with propagated [Ca 2+ ] i rises. Such a transient depolarization probably participated in the occurrence of propagated [Ca 2+ ] i rises as it overlapped with the activation threshold of both Na + and Ca 2+ channel currents. However, we did not observe large spikes that coincided with the spreading of [Ca 2+ ] i waves, possibly because of the poor space-clamp control in the highly coupled FS cells that propagated large-scale [Ca 2+ ] i waves. Consistent with this assumption, it was possible to trigger action potentials in FS cell somas, but only when the cells displayed a high input resistance. Also, the electrical signal might be filtered when passing through gap junctions, as observed between gap junction-coupled neurons [START_REF] Galarreta | [END_REF][START_REF] Venance | Proc. Natl. Acad. Sci. U S A[END_REF]. Hence, techniques that permit sub-cellular monitoring of membrane potential (e.g. potential-sensitive dyes) [START_REF] Yuste | Imaging neurons: a laboratory manual[END_REF] need to be developed for pituitary slices to better address this issue that should include the detection of signals within the long and slender processes of FS cells (16), which are rich in gap junctions (27).

What might be the function of such a network of cells wiring up the anterior pituitary? It has been well documented that FS cells are not just sustentacular elements but perform regulatory functions that determine the responsiveness of hormone secreting cells (14,29). With their long cytoplasmic processes between other cell types they are in an ideal position to play an important role in intercellular communication mechanisms. Although FS cells do not secrete hormones, they are known to release biologically important factors and signaling molecules (e.g. IL-6, nitric oxide, VEGF, bFGF, follistatin) (14,29,[START_REF] Yu | Proc. Natl. Acad. Sci. U S A[END_REF] and are responsive to central and peripheral stimuli (e.g. PACAP, VIP, estrogens) [START_REF] Yada | [END_REF](38)(39) including immune factors (TNFα, TGFß3, interferon-γ) (40)(41)(42). Moreover and most remarkably, the FS cell network seems to provide a unique system of large-scale communication that may rapidly adjust cellular activities within the anterior pituitary. Hence, distant endocrine cells might receive coordinated information from both extrapituitary molecules (1-4) slowly carried by the pituitary microvasculature (5) and intrapituitary signals mostly emanating from the FS cell circuitry.

Legends to figures

  types of action potential were able to induce a transient [Ca 2+ ] i rise when patch-clamp recordings were simultaneously combined with [Ca 2+ ] i imaging (Figs. 2c-d, 10 out of 15 cells).

  since i) it did not alter the initial [Ca 2+ ] i rise in the stimulated FS cell (Fig.4a, middle panel), ii) it markedly decreased the cell-to-cell diffusion of small molecular weight dyes (Neurobiotin TM tracer, 13 fields; β-Ala-Lys-Nε-AMCA, see below), iii) it caused an increase in input resistance in FS cells probably reflecting a reduction of electrotonic coupling (841 ± 152 MΩ versus 1589 ± 250 MΩ in control and carbenoxolone-treated FS cells, respectively, n = 41 and 22, p<0.05), and iv) it also significantly diminished the propagation of intercellular [Ca 2+ ] i waves initiated by Ins(1, 4, 5)-trisphosphate dialysis (40 µM IP 3 in the intra-pipette solution) without suppressing the initial IP 3 calcium-mobilizing response (n = 6 and 11 fields in control and carbenoxolone-treated cells, respectively; p<0.001, chi-square test). Interestingly, IP 3triggered [Ca 2+ ] i waves were much slower to propagate (15 ± 5 µm/s, n = 16 cells) than electrically-induced [Ca 2+ ] i waves (p< 0.05, Mann-Whitney U test). Finally, cell-to-cell transfer of Lucifer yellow that diffuses through most gap junctions revealed that the FS cells undergoing [Ca 2+ ] i waves were dye-coupled (Fig.4b, n = 7 fields).

Figure 1 .

 1 Figure 1. Generation of propagated [Ca 2+ ] i waves in folliculostellate cells in response to electrical stimulation. a, Superimposed DIC image of the slice surface with β-Ala-Lys-Nε-AMCA fluorescence (pseudocolored in blue) of folliculostellate cells (scale bar, 10 µm). b, A confocal image showing ten FS cells loaded with a fluorescent Ca 2+ dye (scale bar, 10 µm). The color circles highlight the area of each cell used to monitor changes in fluorescence reflecting [Ca 2+ ] i levels. The stimulating micropipette was touching the cell circled in green. c, Changes in fluorescence, normalized to baseline fluorescence (F/Fmin), for the ten regions in b. The spread of a [Ca 2+ ] i wave was initiated by a brief electrical stimulation. Onset of the stimulation is indicated by an arrow.

Figure 2 .

 2 Figure 2. Generation of [Ca 2+ ] i transients depends on membrane excitability. a, Stimulated [Ca 2+ ] i transients in two adjoining FS cells (red trace: triggered cell) in normal Ringer's saline (top panel) and in the presence of 0.5 µM TTX (middle panel) are shown. The TTXmediated alteration of [Ca 2+ ] i transients recovered following a 12 min wash (bottom panel). b, Step current injection triggered an all-or-none action potential (black trace) which was suppressed in the presence of 0.5 µM TTX (red trace). c, Current-clamp recording of a single action potential triggered upon current step (bottom trace) that caused a transient rise in [Ca 2+ ] i (top trace). d, Similar combined electrical and optical recordings in a TTX-treated cell.

Figure 3 .

 3 Figure 3. Voltage-gated currents in FS cells. Voltage-clamp recordings were carried out in FS cells (holding potential = -80 mV). a, top panel, Inward current triggered upon step depolarization (black trace). TTX (0.5 µM) blocked the inward current (red trace). Bottom panel, Relative current-voltage relationship of the peak inward current (mean ± SEM). b, top panel, Inward current carried by Ba 2+ ions (in the presence of TTX) (black trace) and triggered upon depolarization to -20 mV. Cd 2+ ions (500 µM) suppressed the Ba 2+ current (red trace). Bottom panel, Relative current-voltage relationship of peak Ba 2+ current. c, Outward currents triggered upon a series of voltage steps (-80 to +80 mV, 20 mV increment). d, Similar recordings in another cell treated with 15 mM TEA.

Figure 4 .

 4 Figure 4. Propagation of [Ca 2+ ] i waves between FS cells depends on gap junctional signaling. a, Electrically-stimulated [Ca 2+ ] i waves between FS cells (red trace: triggered cell) in normal Ringer's saline (top panel) and in the presence of 100 µM carbenoxolone (middle panel) are shown. Carbenoxolone reduced the number of FS cells propagating the

Figure 5 .

 5 Figure 5. Long-distance propagation of electrical and calcium signals along the FS cell circuit. a, Bright-field image of a coronal pituitary slice (x4 objective). The irregular transverse lines were the nylon threads used to keep the slice in place. The two red dots help identify the position of the two stimulation pipette tips touching the slice surface. Scale bar, 500 µm. b, [Ca 2+ ] i changes were recorded (5 frames/s) in five FS cells that were located in the region delimited by the white circle (620 µm from the pair of stimulation pipettes) in a. Electrical stimulation (700 µA, 75 ms) induced the occurrence of coincident [Ca 2+ ] i rises in these cells. c, In another slice, electrical and [Ca 2+ ] i recordings were simultaneously combined in an FS cell, 520 µm away from the point of stimulation. [Ca 2+ ] i rise did not occur, neither did depolarization between the electrical stimulation and the beginning of the plots (39 s time span). Transient depolarizing events then coincided with the propagated [Ca 2+ ] i rises.

Figure 6 .

 6 Figure 6. Three dimensional, large-scale communication through the FS cell network. a, Three-dimensional reconstruction (20-µm-depth) of Neurobiotin TM -filled FS cells (labeled in green) together with the immunolabeling of laminin, a component of basal laminae surrounding the cell cords (labeled in red). The arrow indicates the approximate location of the cell initially recorded during 90 min with a Neurobiotin TM -containing patch-clamp pipette (scale bar, 50 µm). b, FS cell network visualized with the Vaseline-gap technique. Left panel, Coronal slice straddling between two compartments of a chamber. Fluorescent lectin stainings localized the Vaseline bridge covering the slice at the compartment interface (scale bar, 500 µm). Colored dashed lines delineate the approximate location of the Vaseline bridge, while the white one shows the slice periphery. Middle panel, Bath application of β-Ala-Lys-Nε-AMCA in the top compartment allowed the staining of FS cells across the whole slice. Right panel, When carbenoxolone (100 µM) was present in both compartments, β-Ala-Lys-Nε-AMCA-stained FS cells were only seen in the top compartment and, marginally, within the Vaseline gap.

Acknowledgements. We thank A. Carrette and J.-M. Michel for technical assistance.

Supported by INSERM and grants from the European Union, Région Languedoc-Roussillon, Association pour la Recherche sur le Cancer, and Fondation pour la Recherche Médicale.