Topographical anatomy of the albino rat’s ischiotrochanteric muscle group

The obturator internus, gemelli, and quadratus femoris muscles are thought to be postural muscles. Thus, they are in the focus of research. Although these muscles are described in other species, detailed descriptions are still lacking for the albino rat. We hypothesized that the rat’s ischiotrochanteric muscle group is comparable to that of humans. We aimed to provide a detailed description, and to compare the rat’s condition with other species including humans. Both hind limbs of 30 adult male formalin-fixed albino rats were carefully dissected and photo documented. Our results were then compared with data for other species and descriptions of human anatomy. The gemellus muscle was one single muscle mass, originating from the lesser sciatic notch and an unnamed groove on the dorsal aspect of the ischium. The obturator internus muscle arose from the inner aspect of the tabula of ischium. Both muscles formed a continuum and inserted as one complex on the medial aspect of the greater trochanter. The quadratus femoris muscle originated from the outer aspect of the tabula of ischium and inserted on the distal portion of the intertrochanteric crest, and the dorsal aspect of the lesser trochanter. Despite minor differences, the topographical conditions of these muscles are comparable between rats and other mammals including humans.

Phylogenetically and ontogenetically, in all mammals including humans, the ischiotrochanteric muscle group includes the quadratus femoris muscle, the obturator internus muscle, and the gemelli muscles. These muscles are innervated by two nerves arising variably from the sacral plexus, the nerve to quadratus femoris muscle, and the nerve to obturator internus muscle [1][2][3][4][5] . The muscles of the ischiotrochanteric group, together with the piriformis muscle and the obturator externus muscle, form the short lateral (external) rotators of the hip joint, often referred to as the "rotator cuff of the hip joint" 6 . These muscles are thought to be important as postural muscles, more than as prime movers 7 . For this reason, they are also the focus of research [8][9][10][11] . Animal models are often used for this purpose 12,13 .
Although these muscles are partly well described in humans and primates [6][7][8]14,15 , there are no sufficiently precise descriptions for the albino rat, which is after all used as a frequent animal model for basic 16 and translational research 17 . Books on the anatomy of the laboratory rat 18,19 outline this subject only cursorily or erroneously and are therefore neither useful for comparison with other species (including humans) nor for exact dissection. On the other hand, there are no descriptions of rodents even in detailed books of comparative anatomy 4,20 , which would be helpful for exact dissection of the hip and thigh regions in the rat. According to former comparative anatomists, the ischiotrochanteric muscle group is said to hardly vary within mammals, which makes them well comparable between species 4,21 .
We hypothesize that the topography of the rat ischiotrochanteric muscle group is also comparable to that of other mammals including humans. To verify this, the first goal was to provide a complete and accurate description and a topographical dissection guide of the muscles of the ischiotrochanteric group in the albino rat. The second aim was to discuss the similarities and differences of the condition found in albino rats and other mammals, including humans from a pure topographical point of view.

Methods
To clarify the general arrangement of the muscles of the ischiotrochanteric group, both hind limbs of 30 adult (12-14 weeks old) male albino rats (24 Sprague-Dawley, 6 Wistar) have been carefully dissected. The rats had been used for other studies not involving the regions of interest and had been sacrificed before being placed to

Results
The systematic details of the investigated muscles are summarized in Table 1. Within this muscle group, there were no remarkable variations. Only, the exact origin of the nerve to obturator internus muscle was subject to small variations concerning its exact origin. This was due to the somewhat variable formation of the sacral plexus and its branches.
Obturator internus and gemellus muscles-biceps coxae muscle. The obturator internus and gemellus muscles are arranged in series building a common tendon of insertion. The two muscle bellies can be only separated by sharp dissection along their approximate border. Therefore, both muscles together will be termed biceps coxae muscle in this study. The obturator internus muscle forms the intrapelvine portion, and the gemellus muscle the extrapelvine portion of this muscle mass. On the sharp edge of the dorsal border of the ischium caudal to the lesser sciatic notch, they are in close contact. Here, the caudal portion of the obturator internus muscle continues more or less into the caudal portion of the gemellus muscle. Both parts are of approximately the same size, but the gemellus muscle is considerably thicker. From the superficial aspect, the gemellus muscle seems to consist of two separate muscles, a small cranial and a large caudal one. However, on its deep aspect, it is one inseparable individual (Fig. 3).
The obturator internus muscle is a pennate muscle, which narrows towards its tendon of insertion. It originates from the inner aspect of the tabula of ischium as far as the caudal border of the obturator foramen (Figs. 2b and 4a). Thus, it is situated caudally to the obturator membrane. The tendon of insertion of the obturator internus muscle courses through the lesser sciatic notch. There, this tendon is deflected in an angle of approximately 90° (Fig. 4a). Therefore, the large sciatic bursa of obturator internus muscle is situated between the deep aspect of this tendon and the bone. The gemellus muscle, which is also a pennate muscle, arises from the lesser sciatic notch around the tendon and the sciatic bursa of obturator internus muscle, and from an unnamed groove, which is located cranially to the ischial tuber (Figs. 2a,c, 4b,c). This groove is separated from the tabula of ischium by a sharp unnamed ridge, which connects the ischial tuber and the acetabulum (Fig. 1e,h). Just outside the lesser sciatic notch, the gemellus muscle fuses with the tendon of the obturator internus muscle, forming the biceps coxae muscle. The tendon of this biceps coxae muscle reinforces the dorsal part of the hip joint capsule, prior to its insertion on the medial aspect of the greater trochanter (Figs. 2d, 4b,c). The obturator internus muscle is innervated by the nerve to obturator internus muscle. It branches off from the pudendal nerve, one of its main branches, or is a direct branch of the sacral plexus. It enters the obturator internus muscle on its superficial surface by piercing its strong fascia (Fig. 4d). The gemellus muscle is innervated by the nerve to quadratus femoris muscle, which enters the muscle on its deep surface (Fig. 4e).
Quadratus femoris muscle. The quadratus femoris muscle is the thickest of this muscle group. It originates from the outer aspect of the tabula of ischium (Figs. 2a,c, and 5a). It crosses the obturator externus and intermedius muscles and inserts on the distal portion of the intertrochanteric crest, and on the dorsal aspect of the lesser trochanter (Figs. 2d and 5a). The quadratus femoris muscle is innervated by the nerve to quadratus femoris muscle branching off from the sciatic nerve. It enters the muscle on its deep surface (Fig. 5b). Topography and functional aspects. As described above, the obturator internus muscle originates from the inner aspect of the tabula of ischium caudal to the obturator foramen. In the region of the ischial tuber and the ramus of ischium, it is situated adjacent to the origins of the hamstring and adductor muscles. The obturator internus muscle is covered by a strong fascia. This fascia, together with the obturator membrane, is attached to www.nature.com/scientificreports/ the dorsocaudal part of the boundary of the obturator foramen. From the junction of these two fascial structures parts of the pelvic floor arise, forming the tendinous arch of levator ani muscle (Fig. 6a). Dorsal to this tendinous reinforcement, the pudendal canal is located between the external fascia of pelvic diaphragm and the fascia of obturator internus muscle. This canal has a craniocaudal course and contains the two main branches of the pudendal nerve and the accompanying vessels (Fig. 6b). The nerve to obturator internus muscle pierces the fascia of the obturator internus muscle to innervate this muscle (Fig. 4d).
The gemellus muscle arises from the ischium around the lesser sciatic notch and from an unnamed groove cranially to the ischial tuber (Figs. 1e,h, 2a,c). There it contacts the origin of the biceps femoris muscle at the dorso-cranial part of the ischial tuber. Its cranial portion has close contact with the gluteus profundus muscle and is partially covered by it. The caudal portion is in close contact with and partially covers the quadratus femoris muscle (Fig. 6c,d). The tendon of the biceps coxae muscle covers the common tendon of the obturator externus and intermedius muscles from dorsal (Fig. 6d). The belly of the gemellus muscle and the tendon of the biceps coxae muscle are covered by the caudal portion of the gluteus medius muscle, the caudofemoralis muscle and the vertebral head of the semitendinosus muscle. Bony structures important for this study. Macerated bones from a male albino rat were used to provide an overview of the bony structures important for this study. Isolated right femur from (a) the cranial, (b) the caudal, (c) the lateral, and (d) the medial aspect. Isolated right hip bone from (e) the lateral, (f) the medial, (g) the ventral, and (h) the dorsal aspect. a = acetabulum, gsn = greater sciatic notch, gt = greater trochanter, I = ilium, ic = intertrochanteric crest, it = ischial tuber, is = ischial spine, lsn = lesser sciatic notch, lt = lesser trochanter, of = obturator foramen, P = pubis, ri = ramus of ischium, sps = sacropelvic surface (auricular surface and iliac tuberosity), ti = tabula of ischium, tt = third trochanter, ug = unnamed groove dorsal on the ischium, ur = unnamed ridge dorsal on the ischium, asterisk = trochanteric fossa. www.nature.com/scientificreports/ The quadratus femoris muscle originates from the outer surface of the tabula of ischium. There it has close contact with the gemellus, pelvic head of semitendinosus, biceps femoris, semimembranosus and obturator externus muscles that originate around the tabula of ischium. Dorsally, the quadratus femoris muscle is covered proximally by the gemellus and vertebral head of the semitendinosus muscle and subsequently by the caudofemoralis muscle. The ventral portion of the quadratus femoris muscle is flattened near its origin by the course of the large semimembranosus muscle. The adductor minimus muscle superficially crosses the half of the quadratus femoris muscle close to its insertion. The quadratus femoris muscle is oriented nearly horizontally and thus it crosses the obturator externus muscle, which courses diagonally craniodorsal to its insertion in the trochanteric fossa. Therefore, the quadratus femoris muscle has no direct contact with the capsule of the hip joint (Figs. 5a and 6e). In the region of its insertion, the quadratus femoris muscle is in close contact with the insertion of the iliopsoas muscle.
The sciatic nerve runs superficially to the biceps coxae and quadratus femoris muscles. There its tibial part gives off the muscular branch for the hamstring muscles. Still deep to the piriformis muscle, the sciatic nerve gives off the nerve to quadratus femoris. The latter sends a small branch into the caudal portion of the gluteus profundus muscle and then disappears deep to the gemellus muscle (Fig. 6c). There it gives off the innervation for Color key: blue-gemellus muscle, orange-obturator internus muscle, red-tendon of biceps coxae muscle, green-quadratus femoris muscle. Table 1. Systematic anatomy of the muscles of the ischiotrochanteric group in the albino rat. For each muscle, the exact origin, insertion, and innervating nerves are summarized here.

Muscle Origin Insertion Innervation
Obturator internus muscle www.nature.com/scientificreports/ this muscle and finally reaches the deep side of the quadratus femoris muscle (Figs. 4e and 5b). At their origin, the gemellus and quadratus femoris muscles are crossed by the superficial perineal nerve running caudally (Fig. 6c).
Since the rat hip joint is a ball and socket joint, in terms of arthrokinematics, it has three main degrees of freedom. The axes for the main movements are as follows: flexion and extension occur about a transverse axis through the femoral head. Abduction and adduction occur about a sagittal axis through the femoral head. External and internal rotation occurs about a longitudinal axis that passes approximately through the shaft of the femur. Because of the location of the quadratus femoris muscle in the neutral position of the hip joint ( Fig. 6c), it courses dorsally and approximately 90° on the axes of rotation. Therefore, it can powerfully externally rotate the hip joint. In addition, it can participate in adduction of the femur in this joint. The biceps coxae muscle also lies dorsal to the axis of rotation (Fig. 4b,c). It, too, should be able to participate in external rotation. Due to its course in relation to the abduction and adduction axis, it assists in abduction of the hip joint. Because of its position close to the fibrous capsule, it should also be able to center the femoral head in the acetabulum.

Dissection guide.
To gain complete access to the muscles of the ischiotrochanteric group, all superficial muscles should be at least partially removed. In any case, the hamstring muscles and the superficial parts of the gluteal muscles must be detached from their origins. The deeper parts of the gluteal group must be detached from their insertions on and around the greater trochanter. To increase the mobility of the hip joint for further preparation, it is also advisable to release the muscles of the adductor group from their origins.
The muscle belly of the obturator internus muscle can best be reached via the ischiorectal fossa. To do this, one must widen the gap between the fascia of the muscles of the tail and the hip bone. As there is neither a sacrotuberal nor a sacrospinous ligament in the rat, this is easily possible (Fig. 7a). In order to gain better access, the sacroiliac joint can be luxated carefully and the pelvic symphysis can be cut (Figs. 4d and 6a). Thereby, attention must be paid to the sacral plexus and some of its nerves (sciatic nerve, superficial perineal nerve, nerve to obturator internus muscle, and pudendal nerve) (Fig. 6b). The external fascia of pelvic diaphragm becomes visible, and must be carefully removed by blunt dissection. During this step, the pudendal canal, which is covered by this fascia, is opened and the innervation of the obturator internus muscle can be visualized (Fig. 7b). To gain more space, the pelvic floor can be detached from the tendinous arch of levator ani muscle and from the pubis (Figs. 4d and 6a). During this, it is essential to preserve the innervation of the obturator internus muscle and the obturator nerve (from the lumbar plexus) on its course towards the obturator canal (Fig. 6a).
Harvesting the biceps coxae muscle can be done best by first detaching its tendon of insertion from the medial aspect of the greater trochanter. Thereby, one has to take care of the quadratus femoris muscle and the tendon of the obturator externus muscle, as they are situated close to the biceps coxae muscle. The tendon of the biceps coxae muscle can be carefully detached from its attachments and can be mobilized together with the gemellus muscle towards the lesser sciatic notch. Thereby, the gemellus muscle can be peeled off from the bone by blunt dissection. It is firmly fixed only on the dorsal border of the ischium around the lesser sciatic notch, from which it has to be detached by sharp dissection (Fig. 7c). During mobilization of the gemellus muscle, its innervation by a branch of the nerve to quadratus femoris muscle has to be cut through. The tendon of the obturator internus muscle can now be carefully released from the lesser sciatic notch. Thereby, the sciatic bursa of obturator internus muscle between the bone and the deep aspect of the tendon is opened (Fig. 7c). The obturator internus muscle can be carefully peeled out from the concave inner aspect of the tabula of ischium. Only the borders of the muscle have to be sharply detached from the bone (Fig. 7d). Superficially, the gemellus muscle appears to consist of two separate individuals. (b) However, on its deep aspect it becomes clear that it is actually one single muscle, which also forms a continuum with the obturator internus muscle. This whole muscle complex is therefore referred to here as the biceps coxae muscle. g = gemellus muscle, oi = obturator internus muscle. www.nature.com/scientificreports/ The quadratus femoris muscle can be detached starting at its insertion on the caudal aspect of the lesser trochanter and the intertrochanteric crest. There it is in close contact to the iliopsoas muscle whose tendon is The muscles of the gluteal, the hamstring, and the adductor groups (except the obturator externus muscle) are detached. The sacroiliac joint is luxated, and the pelvic symphysis cut to get access into the ischiorectal fossa. bc = tendon of biceps coxae muscle, g = gemellus muscle, GT = greater trochanter, hc = hip joint capsule, I = ilium, IT = ischial tuber, oi = obturator internus muscle, om = obturator membrane, qf = quadratus femoris muscle, 1 = sciatic nerve (always dislocated from its true position to gain a complete view), 2 = nerve to quadratus femoris muscle, 3 = superficial perineal nerve, 4 = branch of pudendal nerve, 5 = nerve to obturator internus muscle. www.nature.com/scientificreports/ attached to the ventral part of the lesser trochanter, which should not be injured when mobilizing the quadratus femoris muscle. The latter can be released from its origin on the outer aspect of the tabula of ischium by blunt dissection; during this, the nerve to quadratus femoris muscle has to be cut. When releasing the quadratus femoris muscle, care must also be taken to the obturator externus muscle, which is situated deep to it.

Discussion
The gemelli muscles and the obturator internus muscle are regarded as three individuals, both in veterinary anatomy 5,22,23,27,28 and in human anatomy 26 . The rats studied here had one single gemellus muscle, which in addition forms one unit with the obturator internus muscle. This complex is called biceps coxae muscle in this study. The gemelli muscles are said to form two individual muscles in humans, monkeys, many rodents, and other species. In prosimians, insectivores and others they are more or less fused or form a common mass 4 . However, it is often described that the two gemelli muscles-at least deep to the tendon of the obturator internus muscle-are actually one entity 6,8,14,20,29 , as was also consistently observed in this study. Furthermore, the obturator internus and gemelli muscles are frequently considered as one single mass 4,6,14,21,29 . Thereby, the gemellus muscle represents the extrapelvic or accessory head of the obturator internus muscle 6,29 or, conversely, the obturator internus muscle forms the middle head of the gemelli muscles, which has migrated into the pelvis 14 . The gemelli muscles form a kind of bed or tunnel for the tendon of the obturator internus muscle 7,14 . These three muscles are therefore often referred to as the triceps coxae muscle in humans, at least because of their common insertion 30,31 . The gemelli muscles are subject to wide variation within humans; they may be doubled or even absent. They can be completely fused with the obturator internus muscle itself but also with the quadratus femoris muscle. The latter can even be completely replaced by the gemellus inferior muscle 32 . However, our long experience in the dissection room shows that these variations are extremely rare. In the rats examined here, the caudal part of the obturator internus muscle continued more or less directly into the caudal part of the gemellus muscle. It therefore makes sense to consider the gemelli muscle as one-the extrapelvic-head and the obturator internus muscle as the other-intrapelvic-head of a common muscle mass. This can also be supported by the phylogeny and ontogeny of this muscle group. The muscles of this group originate from a common muscle mass, the quadratus femoris primordialis of the amphibians (anurans) 21 . Only through further developmental steps does the obturator internus muscle separate together with the gemellus muscle from the later quadratus femoris muscle. The obturator internus muscle then gradually migrates to the wall of the lesser pelvis and the gemellus muscle remains in its original position 3,4,21 , apparently without separating completely. This also explains the variations in the appearance of these muscles described above. Therefore, it makes generally more sense to refer to the gemellus muscle (as one individual) as the extrapelvic and the obturator internus muscle as the intrapelvic part of one singular muscle.
In the rats studied here, the obturator internus muscle constantly arose exclusively from the inner aspect of the tabula of ischium, which has never been described before for this muscle. In other rodents (Cricetidae, Heteromyidae), the obturator internus muscle originates either variably from the bones bordering the obturator foramen and the obturator membrane or also from the tabula of ischium (without mentioning this term). In the latter case, however, it additionally arises from the dorsocaudal half of the obturator membrane 5,27 . Parsons 33,34 studied many different rodents and described the origin of the obturator internus muscle as similar to that of humans, i.e. around the obturator foramen. In rhesus monkeys, it also arises from the inner surface of the pelvis and covers the obturator membrane 8 . In domestic animals, the obturator internus muscle arises around the www.nature.com/scientificreports/ obturator foramen and possibly also from the sacral bone and from the obturator membrane 4,20,35 . In cloven hoofed animals (artiodactyls), it does not course through the lesser sciatic notch as in the others, but through the obturator foramen 4,20 and is innervated by the obturator nerve 20 . Looking at the development of this muscle group (see above), it is very doubtful that it is actually an analogous structure of the obturator internus muscle in these cases. In humans, the obturator internus muscle originates from the inner surface of the ventrolateral pelvic wall, medial and caudal to the obturator foramen, from the medial half of the obturator membrane, and its own fascia. The area of origin is said to extend to the ischial tuberosity and the sacrotuberous ligament 6,7,14,15,29 . It is not subject to much variation; rarely it can be absent or completely divided into two parts 32 . The ischium in the region of the ischial tuber is considerably longer (forming the tabula of ischium) in the rat compared to humans, providing enough space for the entire attachment of the obturator internus muscle. In humans and other species with a less pronounced tabula of ischium, the obturator internus muscle apparently has to attach itself further ventrally in the course of its migration into the pelvis. As observed in this study, the cranial portion of the rat's gemellus muscle originated constantly from the lesser sciatic notch around the tendon and the sciatic bursa of obturator internus muscle. The larger caudal portion arose from an unnamed groove cranial to the ischial tuber. In principle, this is consistent with the information given by other authors, even if they have used different terms. Thus, in domestic animals, the dorsal margin of The tendon of the biceps coxae muscle is detached from its insertion, and the gemellus muscle is peeled off from the bone. Its muscular branch from the nerve to quadratus femoris muscle is cut, view from caudolateral. (d) The gemellus muscle is completely released, and the obturator internus muscle is partly peeled off from its area of origin, view from dorsal. The muscles of the gluteal, the hamstring, and the adductor groups (except the obturator externus muscle) are detached. The sacroiliac joint is luxated, and the pelvic symphysis cut to get access into the ischiorectal fossa. bc = tendon of biceps coxae muscle, g = gemellus muscle, GT = greater trochanter, hc = hip joint capsule, IT = ischial tuber, LT = lesser trochanter, oe = obturator externus muscle, oi = obturator internus muscle, pf = pelvic floor, qf = quadratus femoris muscle, white arrow = pudendal canal, white arrowhead = sciatic bursa of obturator internus muscle, white asterisk = external fascia of pelvic diaphragm, 1 = sciatic nerve (dislocated from its true position to gain a complete view in c and d), 2 = nerve to quadratus femoris muscle, 3 = superficial perineal nerve, 4 = pudendal nerve, 4a and 4b = branches of pudendal nerve, 5 = nerve to obturator internus muscle. www.nature.com/scientificreports/ the ischium 20 or the borders of the lesser sciatic notch 4 are mentioned. In other rodents, the superior gemellus muscle is said to arise from the dorsal surface or margin of the ischium; the inferior gemellus muscle along the "superior ramus of ischium" or the "dorsal ischial margin" until the ischial tuber 5,27,28 . In humans, the gemelli muscles arise from (the dorsal surface of) the ischial spine and (the upper part of) the ischial tuberosity 7,14,29 . According to Yoo et al. 6 they are attached to the margins of the lesser sciatic notch, the medial aspect of the ischial tuberosity, and the sacrotuberous ligament. Although in comparative anatomy the trochanteric fossa is usually given as the insertion of the biceps (triceps) coxae muscle 5,20,27,28,35 , in the rat this tendon is attached to the medial surface of the greater trochanter. This is also how it is usually described in humans [6][7][8]14,29 . In humans, the tendon of the triceps coxae muscle can also unite with that of the piriformis muscle 6,7,14,15,29 . This was never observed in the rats in this study. Walters et al. 36 observed that the tendon of the triceps coxae muscle constantly reinforces the capsule of the hip joint in its posterior part. It is mentioned in Gray's Anatomy 7 that there should be a bursa between the tendon and the capsule. Also in this study, the tendon of the biceps coxae muscle was always attached to the dorsal part of the capsule of the hip joint. Since a small hole was often seen in the capsule of the hip joint after this tendon was detached, a small bursa could also be present here, possibly even communicating with the joint cavity. However, this was not investigated in detail here. Apparently, the common tendon of the biceps (triceps) coxae muscle is likely to reinforce the capsule of the hip joint in a dynamic way in both humans and rats.
According to our findings, the quadratus femoris muscle of the rat is relatively similar to that of other mammals and to that of humans. Interestingly, the differences between the species seem to mainly concern the size of this muscle. Ellenberg 20 described it as a rather weak muscle in domestic animals. Jouffrey 4 and Hepburn 37 , on the other hand, characterized it as generally robust or always well developed and its shape as square or triangular. According to this study, the quadratus femoris muscle is in any case relatively strongly developed in the albino rat. This is likely to be the case in other rodents as well, following Parsons 33,34 . Interestingly, the official term tabula of ischium is never used for the origin of the quadratus femoris muscle. The dorsal and external portions of the ischial tuber and the ramus of ischium are often referred to 4,28 . As further proven in this study, the quadratus femoris muscle inserts on the intertrochanteric crest and on the caudal aspect of the lesser trochanter in the rat. Descriptions by other authors are very similar here. Ellenberg 20 and König 35 , for example, described the caudal surface of the femur near the trochanteric fossa. With Jouffrey 4 , the attachment extends to the lesser trochanter or, rarely, even further distally. Rinker 5 and Stein 27 described only the lesser trochanter as the attachment zone in the rodents they studied. In humans, the quadratus femoris muscle may be completely absent and replaced by a large inferior gemellus or obturator internus muscle in rare cases 7,29,32 . It may also be divided at its insertion 32 . The ventral surface or (the upper part of) the outer surface of the ischial tuberosity is named as its origin 7,14,29 . Its attachment is described somewhat variable 6 . Thus, Henle 29 listed the intertrochanteric crest and the area caudal to it. Frohse and Fränkel 14 , on the other hand, wrote that it is almost never attached to this crest, but is attached approximately in the continuation of the gluteal tuberosity until the tip of the greater trochanter. In Gray's anatomy 7 , a small tubercle on the upper part of the intertrochanteric crest and the bone below is mentioned as its attachment.
The muscles of the ischiotrochanteric group are generally innervated by two nerves, the nerve to obturator internus muscle, and the nerve to quadratus femoris muscle. The two nerves are said to be always connected by extra-and intramuscular anastomoses, and in the rhesus monkey, they occasionally even have a common trunk 8 . In humans, they usually originate from the initial part of the sciatic nerve 38 or are direct branches of the sacral plexus 7,8 . In most domestic animals, both branches originate from the sciatic nerve 20,22 . In the rats of this study, the nerve to quadratus femoris muscle always originated from the initial part of the sciatic nerve, whereas the nerve to obturator internus muscle variably originated from one of the two branches of the pudendal nerve or directly from the sacral plexus. According to Kikuchi 39 , the nerve to obturator internus muscle can be classified into three types with regard to its origin. In humans, the sciatic nerve type is said to be the most common. In rats, dogs, cats and rabbits, however, the pudendal type is said to be the most common, which we were able to confirm for rats in this study. The transitional type is said to be most common in the guinea pig. In humans, the obturator internus muscle is always innervated by the nerve to obturator internus muscle, the quadratus femoris muscle is always innervated by the nerve to quadratus femoris muscle 7,14 . The innervation of the gemelli muscles, on the other hand, is somewhat variable in humans. The superior gemellus muscle is usually innervated by both nerves 6,8 , often only by the nerve to obturator internus muscle 7,8 and very rarely only by the nerve to quadratus femoris muscle 8 . The inferior gemellus muscle is mostly innervated by the nerve to quadratus femoris muscle [6][7][8] and only occasionally in addition by a branch from the nerve to obturator internus muscle 8 . Frohse and Fränkel 14 , who considered the gemelli muscles as one mass, described that it is only innervated by the nerve to obturator internus muscle. In the rhesus monkey, the gemelli muscles are mostly innervated by the nerve to quadratus femoris muscle and only in a quarter of cases the superior gemellus muscle is additionally innervated by the nerve to obturator internus muscle 8 . In the rats of this study, the innervation pattern was constant; the gemellus muscle was always innervated by the nerve to quadratus femoris muscle alone, although it formed a common muscle mass with the obturator internus muscle.
This study confirms the statement that the general gross anatomical arrangement of these muscles varies very little within mammals, which makes them well comparable between species 4,21 . From a macroscopic point of view, the topographical conditions of this muscle group in the rat are, in total, comparable to those of humans, even though the rat is quadrupedal. However, there are some differences in detail, which are summarized here.
Due to the obturator internus muscle originating more caudally in the rat, this muscle lies exclusively in the lateral wall of the ischiorectal fossa. The origin of the pelvic floor muscles completely separates the obturator internus muscle from the wall of the lesser pelvis. Where in humans the obturator internus muscle forms part of the wall of the lesser pelvis, in rats there is only the very thin obturator membrane and, on its outer side, the obturator externus muscle, which partly originates from it. However, this difference only concerns the relative www.nature.com/scientificreports/ topographical position to the pelvic floor muscles and not to the hip joint. In any case, the gemellus muscle in the rat consists only of one muscle belly. In humans, the gemelli muscles are fused, at least in the region of insertion, and thus are also more likely to consist of one mass 6,14,29 . However, this should definitely be investigated more closely in humans. There are no real differences between humans and rats in the quadratus femoris muscle. It has a relatively larger area of origin in the rat due to the greater extension of the widened transition from the ischial tuber to the body of the ischium in the formation of the tabula of ischium. We determined the main functions of the biceps coxae and quadratus femoris muscles using a simple arthrokinematic model. Despite the quadrupedal stance of the rat, our results show that these muscles, situated close to the hip joint fulfill almost the same function in the rat as in humans 7 . Despite the rat's squatting position at rest and in motion, we suggest according to Howell 40 that it should be able to move the hip joint in a similar way to humans. This author further concluded that leverage should therefore be altered primarily in the long doublejointed muscles, which have their insertion much more distally than in humans 40 . The small muscles very close to the hip joint, especially the ischiotrochanteric group, show no striking topographical differences in humans and rats. In both species, straight-line locomotion in the hip joint primarily involves movement in the sagittal plane 7,40 . The abductors and adductors are likely to assist in keeping the leg on course in both species. Presumably, the short lateral rotators of the hip joint have a proprioceptive function rather than being prime movers 7 . The rat also needs proprioceptive control of the hip joint despite its quadruped stance and even more so when climbing. In addition, the rat cannot rotate the foot per se, but this is functionally necessary. Therefore, it must be able to lateral rotate the entire leg in the hip joint 40 .
None of the rats in this study had a sacrotuberal ligament nor a sacrospinous ligament. As far as domestic animals are considered, absence of the sacrospinous ligament has been reported only in cats and dogs 20,35 . The sacrum in these rats was also not as ossified as in other animals or in humans and the individual vertebrae are therefore much more mobile. This is probably due to the very mobile tail of the rat. Ligaments in this area would probably even be a hindrance. The sacrospinous ligament is probably a remnant of the tail musculature 41,42 . As this study has shown, the topographical relationships of the pudendal nerve or its branches to the coccygeal muscle in the rat correspond to those of the pudendal nerve to the sacrospinous ligament in humans. In humans, the nerve runs around the much more pronounced ischial spine around the sacrospinous ligament to enter the pudendal canal. In the rat, its branches run similarly-but somewhat more directly-around the caudal margin of the coccygeal muscle. Apparently, with increasing reduction of the tail and thus of its musculature, the coccygeal muscle-still very strongly developed in the rat-is likely to transform more and more towards the sacrospinous ligament.
This study was limited on the topographical description and comparison of the ischiotrochanteric muscle group. In future, further morphological and functional studies, e.g. analysis of muscle architecture or muscle spindle density, are required to verify the exact function of these muscles.

Conclusion
This is the first study that has dealt in detail with the description of the ischiotrochanteric muscle group of the albino rat. In addition to a detailed description of these muscles and a dissection guide, their arrangement in the rat was compared with that described for other species. It could be shown that the muscles of the ischiotrochanteric group in the albino rat are topographically very comparable to those of other mammals including humans due to their location, course, and innervation.


s2fieldsofstudy: Biology Medicine

text:
Survivin knockdown alleviates pathological hydrostatic pressure-induced bladder smooth muscle cell dysfunction and BOO-induced bladder remodeling via autophagy

Aim: Bladder outlet obstruction (BOO) leads to bladder wall remodeling accompanying the progression from inflammation to fibrosis where pathological hydrostatic pressure (HP)-induced alteration of bladder smooth muscle cells (BSMCs) hypertrophic and excessive extracellular matrix (ECM) deposition play a pivotal role. Recently, we have predicted survivin (BIRC5) as a potential hub gene that might be critical during bladder fibrosis by bioinformatics analyses from rat BOO bladder, but its function during BOO progression remains unknown. Here, we investigated the role of survivin protein on bladder dysfunction of BOO both in vitro and in vivo. Methods: Sprague-Dawley female rats were divided into three groups: control group, BOO group, and BOO followed by the treatment with YM155 group. Bladder morphology and function were evaluated by Masson staining and urodynamic testing. To elucidate the underlying mechanism, hBSMCs were subjected to pathological HP of 200 cm H2O and co-cultured with the presence or absence of survivin siRNA and/or autophagy inhibitor 3-MA. Autophagy was evaluated by the detection of Beclin1 and LC3B-II expression, proliferation was conducted by the EdU analysis and PCNA expression, and fibrosis was assessed by the examination of Col 1 and Fn expression. Results: BOO led to a gradual alteration of hypertrophy and fibrosis of the bladder, and subsequently induced bladder dysfunction accompanied by increased survivin expression, while these histological and function changes were attenuated by the treatment with YM155. HP significantly increased survivin expression, upregulated Col1 and Fn expression, enhanced proliferation, and downregulated autophagy markers, but these changes were partially abolished by survivin siRNA treatment, which was consistent with the results of the BOO rat experiment. In addition, the anti-fibrotic and anti-proliferative effects of the survivin siRNA treatment on hBSMCs were diminished after the inhibition of autophagy by the treatment with 3-MA. Conclusion: In summary, the upregulation of survivin increased cell proliferation and fibrotic protein expression of hBSMC and drove the onset of bladder remodeling through autophagy during BOO. Targeting survivin in pathological hBSMCs could be a promising way to anti-fibrotic therapeutic approach in bladder remodeling secondary to BOO.

Introduction
Bladder outlet obstruction (BOO) is characterized by gradually increased intravesical hydrostatic pressure and low flow micturition pattern, is a prevalent condition among aging males that is primarily caused by benign prostatic hyperplasia (BPH) (Capolicchio et al., 2001;Metcalfe et al., 2010;Chen et al., 2012;Komninos and Mitsogiannis 2014). Given the irreversible bladder remodeling, late-stage BOO characterized by bladder decompensation has poor success rates in improving the voiding symptoms even after surgical treatments, which seriously affects the quality of life of patients and their partners (Komninos and Mitsogiannis 2014). Understanding the underlying mechanisms of obstruction to fibrosis may contribute to identifying new targets for pharmaceutical intervention and ensure that patients who live with a degree of obstruction never progress to the end stages of bladder dysfunction.
As shown in previous studies, the role of cytokines including alpha-blockers, antimuscarinics, and β-Adrenergic receptors during BOO has been well-established (Dale et al., 2014;Gumrah et al., 2017;Yamada et al., 2018). In addition, TGF-β signaling could stimulate fibrotic changes in BSMCs after BOO, and miR-133 could modulate TGF-β-induced BSMC phenotypic changes by targeting CTGF (Duan et al., 2015). Platelet-derived growth factor could induce the proliferation of BSMC and basic fibroblast growth factor could upregulate Col3 and induce the proliferation of BSMC (Imamura et al., 2007;Preis et al., 2015). Hypoxia also affected the BSMC fibrosis, and miR-101 protected hypoxia-induced fibrosis by attenuating TGF-β-smad2/3 signaling (Wang et al., 2019). YAP inhibitor verteporfin improved the pathophysiologic changes of BSMC through the regulation of DNA methylation (Sidler et al., 2018). Moreover, increasing evidence indicated that mechanical stimuli contribute to bladder growth and development; while abnormal mechanical conditions secondary to BOO result in detrusor smooth muscle hypertrophy and extracellular matrix (ECM) accumulation (Ito et al., 2021). Our laboratory demonstrated that intravesical hydrostatic pressure triggered multiple signaling pathways including ERK, NF-κB, PI3K/SGK1, AMPK/mTOR, and so on that were involved in inflammation, dedifferentiation, and fibrosis (Chen et al., 2012;Chen et al., 2015;Liang et al., 2017;Chen et al., 2020a). But the mechano-regulatory processes during BOO remain unknown.
Survivin is the smallest member of the inhibitor of apoptosis protein (IAP) family discovered in 1997 (Li et al., 2021). Recently, emerging evidence has suggested that survivin was highly expressed in most tumor tissues and fibrotic diseases, and its expression was associated with a diverse array of cellular pathological and physiological processes such as differentiation, proliferation, and invasion during scarring, liver fibrosis, and vascular remodeling Wang et al., 2018;Sharma et al., 2021;Ye et al., 2022). Interestingly, our previous study has predicted that survivin was significantly associated with the fibrotic stage of BOO (Di et al., 2022). However, its effects during bladder remodeling remain unclear and require further investigation.
Autophagy is a highly regulated catabolic process that maintains cellular homeostasis in response to various damaged, defective, or unwanted stimuli (Geir et al., 2005;Li et al., 2016;Daniel et al., 2018;Nam et al., 2019). Increasing evidence suggests that autophagy is closely related the bladder remodeling secondary to BOO due to its double-edged sword role in pro-cell survival and the pro-cell death properties . Our previous study has demonstrated that autophagy regulated the biological function of BSMCs under HP (Chen et al., 2020b). The emerging data indicated that survivin negatively modulated autophagy levels in various diseases, which piqued our interest in exploring whether the survivin has a similar role in the bladder remodeling of BOO (Ding et al., 2015;Pavel et al., 2018). We hypothesize that survivin plays a crucial role in the pathological process of bladder remodeling during BOO. In the present study, BOO rats were administered with survivin inhibitor YM155 to demonstrate the effect of survivin. To examine its possible underlying mechanism, cells were subject to HP to simulate the mechanical obstruction microenvironment of BOO. The mechanism in this pathological process provided new ideas for the prevention and targeted therapy of bladder remodeling after BOO.
Frontiers in Cell and Developmental Biology frontiersin.org

Materials and methods
Rat bladder outlet obstruction model and experimental design All animal experiments were reviewed and approved by the West China Hospital Committee on Animal Care. Female rats (220-250 g) underwent surgical operation for the establishment of BOO was similar to our previous study (Liu et al., 2017). The sham-operated group underwent the same procedure but without urethral ligation. BOO rats in the experimental group were administered with survivin inhibitor YM155. The control group was treated with saline. BOO rats were given by gavage orally administered with 10 mg/kg YM155 or saline once other days from the third day after the operation for 6 weeks. After 6 weeks, the urodynamic characteristics were detected. Subsequently, rats were humanely euthanized for the detection of bladder weight, bladder structure, bladder fibrosis, and mucosa-removed for WB.

Urodynamics
Cystometry was performed to evaluate the urodynamic parameters as previously described. Briefly, rats were anesthetized and a 25 G needle connecting to a polyethylene catheter was inserted and fixed in the dome of the bladder. Cystometry was performed through the continuous saline infusion at a rate of 2.4 ml/h. The micturition pressure, baseline pressure, threshold pressure, and micturition interval were recorded to evaluate bladder function.

Histology and immunohistochemistry
After the end of the cystometry, bladders were excised and weighed, subsequently fixed in 10% neutral buffered formalin, embedded in paraffin, and cut into 5 μm sections. Masson's trichrome staining was used to observe tissue fibrosis according to protocol. The blue-stained collagen and red counterstained muscle were analyzed utilizing ImageJ (Fiji) software. Ten randomly chosen areas at ×50 optical magnification in representative portions of each slide were calculated in a blinded fashion with a square micrometer and the mean area was expressed as the relative percent. The mean percent collagen area was defined according to the formula: collagen area/total visual area × 100%. We and other teams all have previously published research articles using these methods . The percentage area was selected in the program and generated automatically for each image. The averages were then calculated for each group. For immunohistochemistry, paraffin sections were processed with deparaffinization, antigen retrieval, removal of endogenous catalase, and blockage of the unspecific binding site. Sections were then incubated with primary antibodies overnight at 4°C. After washing in PBS, anti-mouse AF594 (A11032) and antirabbit AF-488 (A21206) from Invitrogen were used for incubation for 1 h in the dark at room temperature. Finally, nuclei were counterstained with DAPI. The intensities of α-SMA, Col1, and Fn were obtained using ImageJ software and normalized against DAPI intensities.
Cell culture and reagents hBSMCs (No. 4310, ScienCell, San Diego, CA) were cultured with a special smooth muscle cell medium (SMCM) containing 10% FBS and 1% penicillin/streptomycin. Cells between passages 2 and 6 were incubated in conventional culture conditions (5% CO 2 and 37°C) for all experiments. hBSMCs were pretreated with the indicated intervention reagents for 1 h before exposure to HP. The survivin inhibitor YM155(HY-10194) was obtained from MCE and autophagy antagonist 3-methyladenine (3-MA; M9281) was obtained from Sigma-Aldrich (St. Louis, MO).
Hydrostatic pressure hBSMCs were subjected to Hydrostatic pressure (HP) in our designed apparatus, which provided conventional culture conditions (5% CO 2 and 37°C) for cell growth. Similar to the previous report study, hBSMCs in the apparatus were subjected to HP of 200 cm H 2 O for the pathological parameter mimicking the pathological condition of BOO (Chen et al., 2020a).

Microarray data of the obstructed bladder
Microarray analysis (service provided by Kangchen Biotech, Shanghai, China) was based on our previous study to evaluate the bladder gene expression in the BOO model and sham-operated group using a Whole Rat Genome Oligo Microarray (Agilent Technologies, Santa Clara, CA) (Yang et al., 2018). After the microarray data of the BOO and sham-operated rats were obtained, we transformed the probe identification numbers into gene symbols. Moreover, we normalized the gene expression values with the Affy package, and log2 transformation and normalization were applied in the Robust Multichip Average signal intensity analysis. Our previous study predicted the potential role of survivin during BOO fibrosis by bioinformatics analyses. To investigate the specificity of survivin in BOO bladder, we further compared all IAP family member's expression both in mRNA level and protein level between BOO bladder and sham-operated bladder, and we finally confirmed that only survivin expression significantly increased in BOO bladder compared with shamoperated bladder.

Statistical analysis
Three independent observations for each in vitro cellular experiment have been performed (n = 3) and in vivo rat experiments were divided into three groups (n = 8). Statistical analyses were performed with Graph Pad Prism 8 and with SPSS 20.0 software. The data were shown as the mean ± SD. D'Agostino-Pearson normality test was used to evaluate whether the continuous data were normally distributed, and according to the results, Student's t-test or ANOVA for normally distributed data or a Kruskal-Wallis 1way ANOVA test for not normally distributed data was used.
A p-value less than 0.05 was considered statistically significant.

Bladder outlet obstruction-induced the upregulation of survivin expression
The underlying mechanism of bladder fibrosis flowed by BOO was not well known. Our microarray analysis results showed that among the common IAP members in the bladder, BIRC3 and survivin (BIRC5) were prominently upregulated in the BOO rat bladder when compared with those in the sham-operated bladder, while others were not changed significantly ( Figure 1A). However, to better understand the role of survivin in bladder smooth muscle, the bladder-removed mucosa was used to assess the protein expression of survivin and BIRC3 by Western blot. As shown in Figure 1B, the survivin expression of mucosa-denuded bladders in BOO bladder was significantly increased when compared with sham-operated bladder. However, there was no significant difference in BIRC3 protein expression between the sham-operated group and the BOO group.

The effect of YM155 on bladder histology alteration secondary to bladder outlet obstruction
Our previous study has predicted that survivin was a critical hub gene in bladder fibrosis (Di et al., 2022). To investigate the effect of survivin during bladder remodeling, the rats were administered survivin inhibitor YM155. Based on the results of urodynamic parameters and Masson staining, BOO was successfully established after obstruction for 6 weeks. As we know, BOO significantly induced the deposition of ECM. After the performance of urodynamic testing, bladders were harvested for the evaluation of bladder weight, histological alteration, and collagen deposition. The results showed that the bladder weights of BOO significantly increased compared with the sham-operated group and the increase was attenuated by the treatment with YM155 ( Figure 2A). Nevertheless, body weight did not display evident changes ( Figure 2B). We observed the survivin protein in the BOO bladder decreased after the treatment with YM155. Similar trends were observed in the protein levels of PCNA ( Figure 2C). Based on Masson's trichrome staining results, BOO markedly increased the percentage of collagen in the bladder, while treatment with YM155 decreased the percentage of collagen ( Figure 2D). As we know, bladder remodeling characterized ECM deposition mainly including Col1 and Fn. As Figure 2E showed, we further found that YM155 mainly decreased the Col1 and Fn Frontiers in Cell and Developmental Biology frontiersin.org of the bladder by the immunohistochemical evaluation ( Figures  2F,G). These results indicated that YM155 might reverse bladder remodeling secondary to BOO.

The effect of YM155 on bladder outlet obstruction-induced bladder dysfunction
The bladder function was analyzed according to the urodynamic curve. As shown in Figures 3A-C, the micturition interval in the BOO group was significantly shorter than in the sham-operated group, while treatment with YM155 attenuated the alteration. Micturition pressure, baseline pressure, and threshold pressure were higher in the BOO group compared with the sham-operated group. Baseline pressure was a slight, but significant, increase in BOO treated with YM155 compared with the BOO group, while micturition pressure and threshold pressure have no significant alteration by treatment with YM155 ( Figures 3D-G). These observations indicated that the YM155 markedly suppressed the dysuria associated with BOO.

The upregulated survivin enhanced the collagen expression and cell proliferation of hBSMC
As our previous study demonstrated, pressure at 200 cm H 2 O was selected as the optimal pathological parameter mimicking the BOO cell model in vitro (Chen et al., 2020a, Jin et al., 2020Gao et al., 2021). Exposure to 200 cm H 2 O HP for 24 h increased the expression of collagen including Col1 and Fn protein levels. In addition, the analysis of EdU and PCNA expression levels showed that HP enhanced the proliferation as well as upregulated survivin. To explore whether the HP-increased survivin was necessary for fibrosis, hBSMCs were transfected with siRNA before exposure to HP. We first confirmed the successful knockdown of siRNA by the western blot ( Figure 4A). As the results of WB showed, survivin siRNA significantly suppressed the HP-induced Col1 and Fn expression ( Figures 4B,C). As the results of EdU and the PCNA expression showed, survivin siRNA repressed the HP-induced cell proliferation ( Figures 4C,D).

Hydrostatic pressure-increased survivin leads to proliferative and fibrotic response through autophagy
Our previous study has demonstrated that autophagy was strongly involved in the function of hBSMCs under HP (Chen et al., 2020b). The emerging data point to several aspects of autophagy that was closely related to survivin protein. To understand whether and how survivin affected autophagy Frontiers in Cell and Developmental Biology frontiersin.org during BOO, autophagy markers in BOO bladder were detected after the treatment with YM155 in vivo. We observed the YM155 alleviated BOO-decreased LC3B II/I and Beclin1 ( Figure 5A). Cells were exposed to HP, and the level of LC3B II/I and Beclin1 were measured after the treatment with siRNA survivin in the presence or absence of 3-MA. As shown in Figure 5B, siRNA survivin treatment promoted autophagy level, which was evidenced by increased LC3B II and Beclin1, suggesting survivin negatively regulated autophagy level under HP. To investigate whether the increase LC3B II was due to enhanced autophagy or blockage of the lysosomal degradation of autophagic vacuoles, autophagic flux was assessed using the GFP-mRFP-LC3B assay. The results showed that the amounts of autophagosomes and autolysosomes were both increased, indicating that autophagic flux was unobstructed ( Figure 5C). We further found that the autophagy inhibitor, 3-MA, not only decreased the siRNA survivin-increased autophagy level but also increased the siRNA survivin-decreased Col1 and Fn ( Figure 5D). The PCNA expression and EdU analysis also demonstrated that 3-MA ameliorated the siRNA survivinincreased proliferation (Figures 5D,E). These results suggested that survivin negatively regulated autophagy, which played a critical role in fibrosis and proliferation of hBSMCs.

Discussion
Accumulating evidence suggests that the impairment of bladder morphology and bladder function secondary to BOO is associated with fibrosis, which is characterized by bladder smooth muscle hypertrophy and ECM deposition (Uvelius, Persson, and Mattiasson 1984;Metcalfe et al., 2010;Metcalfe et al., 2010). There is widespread consensus that ECM deposition in the detrusor layer is the primary reason for dysfunction. Understanding the underlying mechanism from obstruction to hypertrophy and fibrosis using rat models may contribute to identifying targets for pharmaceutical intervention at the appropriate stage of bladder remodeling.
Emerging studies indicate that survivin is critically involved in various physiological and pathological processes, such as wound healing, neovascularization, scar formation process, and so on, due to its dual effects of promoting the cell cycle  Frontiers in Cell and Developmental Biology frontiersin.org 08 Frontiers in Cell and Developmental Biology frontiersin.org 09 and inhibiting apoptosis (Ding et al., 2015;Miyamoto et al., 2021;Sharma et al., 2021). Our previous report predicted that survivin was well related to bladder fibrosis by bioinformatics analyses from the whole bladder. To better understand the role of survivin in bladder smooth muscle, the bladder wall tested in the present study was removed mucosa. We demonstrated that among IAP members, BIRC3 and survivin were prominently upregulated. Based on the potential role in bladder fibrosis, it was of great interest to explore the effect of survivin on the pathological process of BOO. Altered ECM composition, especially, Col1 and Fn, has been reported in the hypertrophic bladder during the fibrotic stage of hypertrophy. The result showed that targeting inhibition of survivin by YM155 could downregulate the ECM deposition and improve bladder dysfunction secondary to BOO.
The bladder detrusor, which is mainly composed of smooth muscle cells, is considered to be the important structural unit of the bladder wall. The late stage of BOO is characterized by muscle hypertrophy and enhanced production of extracellular matrix constituents. Besides the BSMC secreted collagens, other mechanisms such as epithelial-mesenchymal transition (EMT), fibroblast-tomyofibroblast transition (FMT), and macrophagemyofibroblast transition (MMT) are all involved in bladder fibrosis. Although collagen that existed in the bladder detrusor is not the most, the collagens in intercellular spaces of smooth muscle cells seriously affect bladder function. The biomechanical force caused by BOO is a critical element for bladder smooth muscle cell function and phenotype. Therefore, the present study focused on the bladder smooth muscle cells. The persistent obstruction increased intravesical pressures, which played a critical role in bladder remodeling (Chen et al., 2012;Liang et al., 2017). To extensively illustrate the underlying mechanism, hBSMCs were exposed to the HP of 200 cm H 2 O mimicking the conditions of BOO, and were used as the experimental model for the present investigation. We found that HP of 200 cm H 2 O significantly increased collagen expression and enhanced cell proliferation as well as upregulated survivin, which was similar to the results in vivo. We further used siRNA to knock down survivin protein, the results showed that siRNA survivin ameliorated the HPincreased fibrotic and proliferative response.
As a multifunctional factor widely existing in mammals, autophagy participates in various physiological and pathological processes, such as immunity, inflammation, fibrosis, and so on. Our previous study indicated that HP significantly enhanced the proliferation and contraction of BSMCs, and we found that autophagy was of importance to BSMCs dysfunction. While the underlying mechanism is not well known. Emerging evidence suggests that crosstalk between apoptosis and autophagy plays an important role in supporting cell survival and proliferation. Survivin, as a member of the IAP family, was initially thought to be a connection with autophagy. We firstly indicated that survivin knockdown abolished the HP-decreased autophagy level by assessing the LC3B II/I ratio and Beclin1 expression. Subsequent experiments further revealed that autophagy inhibitor 3-MA suppressed the survivin knockdown-induced changes.
This present study still has some limitations. Firstly, our study failed to explore human BOO tissues or samples for ethical reasons. Secondly, the effect of YM155 on HP-induced fibrosis of hBSMCs needs to be further explored. In conclusion, HP-induced survivin negatively regulated autophagy, which played a critical role in the pro-fibrosis and pro-proliferative of hBSMCs in vitro. A novel antifibrotic and anti-proliferative function of survivin might represent a potential target for therapeutic strategies in bladder remodeling. Clinical trials were needed to validate the effect of survivin on bladder dysfunction and bladder fibrosis in the future.

Data availability statement
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.

Ethics statement
The animal study was reviewed and approved by the West China Hospital Committee on Animal.

Author contributions
GC and LY conceived the research. GC, YL(8th author), and SC have contributed to the conception and design of the study. YL(5th author), RQ, SH, and YL(7th author) performed the experiments. LY, YL(8th author), XD, and GC analyzed the data.


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Transcriptome Analysis Identifies Accumulation of Natural Killer Cells with Enhanced Lymphotoxin-β Expression during Glioblastoma Progression

Simple Summary Natural killer (NK) cells play a pivotal role in anti-cancer immunity. We conducted an integrated analysis of single-cell RNA-sequencing datasets of malignant brain tumor-associated immune cells from a mouse glioma model and human newly diagnosed and recurrent glioblastomas. We found a robust increase in NK cells during brain tumor production. The cells showed a gene expression phenotype associated with NK cell dysfunction. Notably, the cytokine Lymphotoxin-β (LTB) was highly expressed in these cells. LTB receptors are found on myeloid and structural cells. In summary, we characterize a dysfunctional NK cell phenotype in advanced glioma. Abstract Glioblastomas are the most common primary brain tumors. Despite extensive clinical and molecular insights into these tumors, the prognosis remains dismal. While targeted immunotherapies have shown remarkable success across different non-brain tumor entities, they failed to show efficacy in glioblastomas. These failures prompted the field to reassess the idiosyncrasies of the glioblastoma microenvironment. Several high-dimensional single-cell RNA sequencing studies generated remarkable findings about glioblastoma-associated immune cells. To build on the collective strength of these studies, we integrated several murine and human datasets that profiled glioblastoma-associated immune cells at different time points. We integrated these datasets and utilized state-of-the-art algorithms to investigate them in a hypothesis-free, purely exploratory approach. We identified a robust accumulation of a natural killer cell subset that was characterized by a downregulation of activation-associated genes with a concomitant upregulation of apoptosis genes. In both species, we found a robust upregulation of the Lymphotoxin-β gene, a cytokine from the TNF superfamily and a key factor for the development of adaptive immunity. Further validation analyses uncovered a correlation of lymphotoxin signaling with mesenchymal-like glioblastoma regions in situ and in TCGA and CGGA glioblastoma cohorts. In summary, we identify lymphotoxin signaling as a potential therapeutic target in glioblastoma-associated natural killer cells.

Introduction
Glioblastomas are aggressive primary brain tumors with a median survival of 14.6 months despite standard-of-care treatment [1]. Evidence from single-cell RNA sequencing studies suggests that glioblastomas and other gliomas arise from a transformation event of glial progenitor cells [2,3]. In line with this origin, glioblastoma cells express astrocyte-like (AC)-like, neural progenitor cell (NPC)-like, oligodendrocyte progenitor (OPC)-like, mesenchymal (MES)-like and other transcriptional programs [2,4,5]. These states are concomitantly found within the same tumor, affecting different functional and prognostic aspects of glioblastoma biology. Recent evidence suggests that neuroectodermal-like tumor cells, i.e., NPC-, OPC-and AC-like cells, engage in crosstalk with resident brain cells, driving diffuse tumor infiltration, while MES-like regions are found at the tumor core [6]. Different modes of crosstalk between neoplastic and resident cells have been described, including microtubules, synapse formation and secretion of soluble factors [6][7][8][9]. MES-like regions are characterized by immune cell infiltrates, hypoxia and genomic instability [2,10]. Integration of molecular and clinical data has associated MES-like regions with aggressive clinical behavior [11,12]. The influences of chronic inflammation and hypoxia-driven genomic instability and epigenomic reprogramming on tumor evolution within MES-like regions are an area of active research [6,13]. Herein, immune cells, a major component of the glioblastoma microenvironment [14], are of particular interest as vectors of targeted immunotherapies.
Several single-cell RNA sequencing studies have examined the myeloid and T lymphoid compartments that represent the most common immune cells in glioblastomas. The complex interactions between T cells, myeloid cells, non-neoplastic astrocytes and glioma cells have recently been highlighted as drivers of the immunosuppressive glioblastoma microenvironment [15][16][17][18][19]. It was shown that brain resident microglia are gradually replaced during tumor progression in humans and mice [20,21]. While glioma-associated macrophages are largely characterized by cytokine/chemokine, antigen presentation, angiogenesis and hypoxia-associated signaling [20][21][22][23], glioma-associated dendritic cells (DCs) show a remarkable heterogeneity with hallmarks of impaired function [20,24]. T cells display the molecular hallmarks of anergy and exhaustion [25]. A recent study found an unexpected expression of an NK cell-like transcription program in glioblastoma-associated cytotoxic T cells highlighting commonalities between adaptive and innate immunity [26]. Conversely, the functional parallels between NK cells and cytotoxic T cells are wellestablished [27].
NK cells are innate lymphoid cells with a central role in anticancer and antiviral immunity [28]. Although they are relatively sparse in glioblastomas [26], several NK-cells targeting tumor immune evasion mechanisms have been identified. Different mechanisms are involved, including integrin and NKG2D ligand binding [29][30][31]. Here, we show that glioma progression is associated with an NK cell transcriptional phenotype characterized by reduced activation, an upregulation of apoptosis genes and enhanced lymphotoxin signaling.
We have integrated single-cell RNA sequencing datasets from GL261 syngeneic glioma models that were obtained 14, 21 and 28 days after injection [20,21,32]. The NK cells showed a robust correlation with the time lapsed after tumor injection. The pseudotime analysis revealed a downregulation of interferon genes and an upregulation of Lymphotoxin-β (LTB), a member of the TNF superfamily involved in the development of adaptive immunity and inflammation [33]. LTB is a ligand to Lymphotoxin-β receptors (encoded by the LTBR gene). LTB is expressed by activated T, B and NK cells as well as innate lymphoid cells, while LTBR is expressed on stromal and myeloid cells [33]. Crucially, LTB-LTBR signaling is required for NK cell development [34]. The connection between LTBR signaling and malignancies is well-established [35]. The gene regulatory network and pathway enrichment analysis showed a downregulation of interferon-and an upregulation of apoptosis-associated genes. We confirmed these findings in humans showing that LTB gene expression was spatially correlated with the NK cell exhaustion marker killer cell lectin-like receptor G1 (KLRG1) [36][37][38]. Mining of the Cancer Genome Atlas Consortium (TCGA) and the Chinese Glioma Genome Atlas (CGGA) glioblastoma cohorts showed enhanced LTB expression in MES-like glioblastomas and an association of LTB expression levels with overall survival. In summary, a crosstalk between the lymphoid, tumor and myeloid cells within a glioblastoma drives the chronic inflammation associated with reduced NK cell function.

Data Processing and Analysis
The count data were downloaded from the indicated sources and loaded into R using the Read10x function of the Seurat v4 algorithm [39]. Doublet exclusion was done using the scDblFinder R package v1.10.0 (https://bioconductor.org/packages/release/bioc/html/ scDblFinder.html accessed on 15 August 2022). The CITE-Seq mouse dataset from [20] was used as a reference for the multimodal reference mapping workflow specified by the following vignette: https://satijalab.org/seurat/articles/multimodal_reference_mapping. html accessed on 15 August 2022. The remaining mouse datasets were integrated using this workflow with default parameters. The human dataset was integrated using the harmony package with default parameters [40].
The NK cell counts were obtained by assessing the relative abundance of NK cells across all cells and biological replicates. Pearson's correlation analysis was conducted on each cell type using the stat_cor function of the ggpubr v0.4.0 (https://cran.r-project.org/ web/packages/ggpubr/index.html accessed on 15 August 2022).
The latent factors underlying the changes in NK cells were identified using the MOFA+ algorithm [41]. The "integration of a time-course single-cell RNA-seq dataset" workflow was used with default parameters as specified under the following vignette: https://raw. githack.com/bioFAM/MOFA2_tutorials/master/R_tutorials/scRNA_gastrulation.html accessed on 15 August 2022.
The pathway enrichment analysis was conducted using the enrichR web interface [42]. Gene lists from the factors identified using MOFA+ were individually loaded into the website, and the top factors were visualized.
The enrichment of time points in each cluster was assessed using the hypergeometric function with Benjamini-Hochberg adjustment for multiple testing.
The cell-cell interaction analysis was run using default parameters as specified by the developers. First, human orthologues of the mouse genes were identified using the biomaRt v2.52.0. Then, the counts and relevant metadata were exported, and the analysis was run in the command line as specified in the readme file under: https://github.com/ ventolab/CellphoneDB accessed on 15 August 2022.
The gene ontology enrichment analysis was conducted using the clusterProfiler v4.4.4. R package [46]. First, the gene names were converted into ENTREZ using the bitr function. Then, the enrichGO function was run.
The spatial co-expression analysis was conducted using the functionality of the SPATA2 v0.1.0. R package. Herein, normalized transcript counts were correlated across 10X Visium spots. Similarly, the spatially weighted correlation analysis of previously defined glioblastoma gene expression signatures with the LTB and LTBR genes was conducted as previously described [10].
Human Protein Atlas image selection and processing: Images regions were obtained from https://www.proteinatlas.org/ENSG00000111321-LTBR/pathology/glioma#img accessed on 15 August 2022. The images were processed in Photoshop 2022 with selective sharpening and increases in vibrancy, brightness and contrast.

The Mouse Glioma Model GL261 Shows a Diverse Immune Cell Compartment with Changes throughout Tumor Progression
We set out to study the longitudinal immune composition of GL261 syngeneic gliomas and validate our findings in human glioblastomas ( Figure 1a). In recent years, several studies have profiled the immune compartments in these mice [17,20,21,32,49]. Interestingly, three recent studies with comparatively large immune cell amounts have collected glioma tissues for single-cell RNA sequencing after 14, 21 and 28 days (14 d, 21 d and 28 d) with minor adjustments of the respective protocols [20,21,32]. We utilized the Seurat singlecell RNA-sequencing analysis workflow with reference-based mapping of the datasets from the three abovementioned time points onto a reference dataset containing single-cell transcript and epitope information [39]. We transferred the cell type labels of the reference dataset to be consistent with the integrated dataset [20]. Visual inspection of the UMAP embedding of the integrated dataset confirmed the mixing of the datasets (Figure 1b). Graph-based clustering showed the presence of different cell states in most of the identified cell types [50] (Figure 1c,d). Cluster marker genes were consistent with the published cell type-enriched genes, including the homeostatic microglia clusters 0, 1, 4, 6 and 22 (P2ry12 and Hexb), tumor-associated microglia clusters 2, 8, 9, 10 (Cd74 and Apoe), monocyte and macrophage clusters 3, 8, 11 (Ly6c2 and Tgfbi), T cell clusters 5, 13, 18, 19, 21 (Cd3d and Trbc2) and NK cluster 7 (Ncr1 and Klra4) (Figure 1e). In order to assess changes in the cell type composition between 14 d and 28 d after GL261 cell inoculation, we conducted a linear regression analysis for the percentages of the respective cell types over the three post-inoculation (p.i.) time points (Figure 2a). Some cell types showed shifts in abundance without reaching a statistical significance of 0.05, e.g., the homeostatic microglia subset TAM1 reducing over time, or cDC2 and mDC increasing over time (Figure 2a). NK cells showed a statistically significant longitudinal increase from 1.26% ± 1.37 standard error of the mean (s.e.m) at 14 d to 5.21% ± 0.857 at 21 d to 8.44% ± 3.95 at 28 d ( Figure 2a). To assess the transcriptional heterogeneity in NK cells across the p.i. time points, we conducted an unsupervised Multi-Omics Factor Analysis v2 (MOFA+) analysis of these cells followed by a pathway enrichment analysis using the enrichR algorithm [41,42]. MOFA+ infers a low-dimensional data representation and returns latent factors that account for variability in single-cell data [40]. Our analysis of the three p.i. time points returned eight latent factors ( Figure 2b). Interestingly, Factor 1, which accounted for the most variability in the data, was mainly represented at 21 d and 28 d, while Factor 3 was mainly present at 14 d and 21 d, and Factor 5 had a gradual increase from 14 d to 28 d (Figure 2b). The enrichR-based analysis of the MSigDB Hallmark 2020 pathways showed myc-, E2F-and mitosis-related pathways among the top five pathways in Factor 1 ( Figure 2c). Interestingly, Factor 3, which was downregulated at 28 d, contained interferon signaling among the top five pathways, while Factor 5 included hypoxia and glycolysis among its top five pathways (Figure 2c). In summary, an exploratory approach to profile immune cells during glioma progression showed a robust time-dependent increase in NK cells that was associated with a timedependent downregulation of interferon signaling and upregulation of hypoxia pathways.

The Mouse Glioma Model GL261 Shows a Time-Dependent Accumulation of NK Cells with Downregulation of Activation Markers and Enhanced Lymphotoxin-β Expression
Having identified NK cells as a highly variable immune cell type during glioma pro-

The Mouse Glioma Model GL261 Shows a Time-Dependent Accumulation of NK Cells with Downregulation of Activation Markers and Enhanced Lymphotoxin-β Expression
Having identified NK cells as a highly variable immune cell type during glioma progression, we sought to characterize the transcriptional heterogeneity within each time point. To this end, we sub-clustered the NK cells and identified seven clusters (Figure 3a). A time-dependent analysis of the cluster composition at each time point showed a relative decline in Cluster 0 and a relative increase in Cluster 1 throughout glioma progression (Figure 3b). An enrichment analysis using hypergeometric testing showed statistically significant enrichment of Clusters 0 and 5 at 14 d, Clusters 2, 3 and 4 at 21 d and Clusters 1, 5 and 6 at 28 d. To assess stepwise gene expression changes, we applied the pseudotime analysis algorithm StemID [42]. This algorithm identified several trajectories (Figure 3c). We assessed the origin and direction of the trajectory based on the differences in transcriptional entropy. Following the notion that clusters with higher entropy represent transcriptional precursor states [51], we identified Cluster 0 as the earliest transcriptional state followed by Clusters 1 and 2 (Figure 3c,d). The heatmap visualization of the smoothed transcriptional changes along the identified trajectory showed a gradual downregulation of Gzma, the interferon signaling-associated genes Irf7, Isg15 and Stat1 and the chemokine gene Ccl5 with a concomitant upregulation of Gzmc and Ltb (Figure 3e). Alterations of interferon genes and Ccl5 expression were previously associated with NK cell exhaustion [38,52]. The TNF superfamily member gene LTB has pleiotropic functions, including cancer inflammation, tertiary lymphoid structure and NK cell development [33][34][35]53]. A visualization of the stepwise cluster composition of the trajectory showed that the second half of the trajectory was almost exclusively composed of cells from 21 d and 28 d (Figure 3e). Representative gene expression UMAP plots confirmed the gene expression patterns of Gzma, Gzmc and Ltb along the trajectory (Figure 3f). Cells from clusters other than Cluster 6 showed expression of the NK marker Ncr1/Nkp46, while Cluster 6 showed expression of T cell markers, including Cd3e and Trac. These cells may represent NKT cells or a recently identified subset of glioma-associated T cells expressing NK cell markers [26]. The enrichment of Cd3e and Trac in cluster 6 suggests that the remaining clusters consist of bona fide NK cells. The gene ontology analysis indicated that Cluster 0's cells were associated with the terms accompanying iron homeostasis, commonly upregulated in the hypoxic tumor environment [54] (Supplementary Figure S1a). Cluster 1 showed expression of inflammation and extracellular matrix-associated terms, and Cluster 2 was associated with protein and RNA homeostasis-related terms (Supplementary Figure S1a). To identify which gene regulatory elements were driving the identified transcriptional changes, we conducted a SCENIC analysis [45]. Interestingly, we identified a downregulation of type I (IRF7) and type II interferon (STAT1) and NF-κB (KLF2) signaling (Supplementary Figure S1b). Finally, protein validation based on CITE-Seq data from 21 d cells [20] showed a downregulation of several NK cell activation markers (Ly49D, CD69) along the trajectory between Clusters 0, 1 and 2 ( Figure 3g). Interestingly, Cluster 2 showed that upregulations of CD117/c-kit and LTBR signaling have been implicated in lymphoid tissue development and tertiary lymphoid structures during development and in cancer [53,55]. The upregulation of CD117/c-kit and LTB suggest an NK cell subset with a dysfunctional/de-differentiated transcriptional state [34]. In summary, we identified an NK cell subset associated with glioma progression. These cells showed a downregulation of NK activation markers and interferon signaling-associated genes, suggesting a dysfunctional NK transcriptional cell state.

Myeloid Cells Are the Main Putative Ligands for Lymphotoxin-β
LTB signaling occurs via a membrane-bound (LTα1LTβ2) heterotrimer binding to LTBR [33]. While LTB is mainly expressed on lymphoid cells, LTBR is found on stromal and myeloid cells. We utilized the cell-cell interaction analysis algorithm CellphoneDB to

Myeloid Cells Are the Main Putative Ligands for Lymphotoxin-β
LTB signaling occurs via a membrane-bound (LTα 1 LTβ 2 ) heterotrimer binding to LTBR [33]. While LTB is mainly expressed on lymphoid cells, LTBR is found on stromal and myeloid cells. We utilized the cell-cell interaction analysis algorithm CellphoneDB to explore this crosstalk across immune cell subsets [56]. The analysis suggested several putative interactions across the immune cell types at the available p.i. time points (Supplementary Figure S2a). We found several T cell subsets involved in putative LTB-LTBR crosstalk (Supplementary Figure S2b). Interestingly, NK cells and cDC2 showed higher expression of LTB between 14 d and 21 d, while neutrophils showed a downregulation of LTB between these time points. Lta was expressed by regulatory and effector T cells with the mean Lta expression increasing between 14 d and 21 d (Supplementary Figure S2c). The 28 d day time point was underrepresented in this analysis, which may be due to the technical differences of this dataset, as the number of the identified interactions in this dataset overall is lower (Supplementary Figure S2a). In summary, during glioma progression, NK cells appear to be increasingly involved in LTB-mediated crosstalk with myeloid cells and likely other non-immune cells that are not present in this dataset.

Human Glioblastoma Progression Is Associated with Dysfunctional NK Cell Accumulation and Enhanced Lymphotoxin-β Expression
To validate our findings in humans, we turned to a dataset of immune cells FACSsorted from newly diagnosed (ND) and recurrent (R) glioblastomas [20]. This dataset contained tumor cells along with diverse immune cell populations (Figure 4a). Notably, we found a significantly higher relative numeric abundance of NK cells in R glioblastomas compared to ND ones (Figure 4b). Apart from higher numeric abundance, cells from R glioblastomas showed significantly higher transcript abundances of LTB compared to their ND glioblastoma-associated counterparts (Figure 4c). Likewise, LTB expression across the immune cell types was enhanced in R glioblastomas (Figure 4d). Next, we conducted a MOFA+ analysis of the latent factors underlying the transcriptional phenotypes of NDand R-glioblastoma-associated NK cells followed by an enrichR-based pathway enrichment analysis. We found a downregulation of the factors associated with the cell cycle (Factors 1, 3, 5, 6) and interferon-gamma signaling (Factor 5) and an upregulation of the factors associated with apoptosis (Factors 2 and 8) (Figure 4e). Next, we sub-clustered the NK cells to assess the stepwise transcriptional changes (Figure 4f). StemID suggested a trajectory based on transcriptional similarity and entropy from Cluster 3 over Clusters 0 to 1 (Figure 4g). Clusters 0 and 1 almost exclusively consist of R-glioblastoma-associated NK cells (Figure 4h). Along the trajectory, we found a downregulation of ISG15, CD69 and CCL5 and an upregulation of LTB, GZMB and FTL (Figure 4h,i). Interestingly, we found an upregulation of KLRG1, a marker gene for exhausted NK cells [36,37] (Figure 4i). A gene ontology analysis showed enrichment of the cytokine and chemokine function-associated terms in Cluster 0 and the RNA and protein turnover-associated terms in Cluster 1 (Supplementary Figure S3a). The SCENIC analysis identified a reduction in gene expression under the control of STAT1, IRF1 and IRF7 between Clusters 0 and 1 (Supplementary Figure S3b). In summary, single-cell RNA-sequencing data from human ND and R glioblastomas showed an accumulation of NK cells with reduced expression of activation, interferon-gamma and cell cycle genes and enhanced expression LTB and apoptosis-associated genes, suggesting an exhausted NK cell phenotype. NK cell exhaustion has been associated with the loss of cell proliferation markers, reduced interferon signaling and apoptosis signaling [38]. Strikingly, the data showed broad similarities with the mouse data above.

LTB-LTBR Crosstalk Is Associated with MES-like Regions of Glioblastomas
Next, we utilized a spatial transcriptomics dataset of human glioblastomas to map LTB and LTBR expression in situ [10]. We found that transcripts of LTB and its receptor LTBR are found in adjacent tumor regions with overlaps at the intercept of these regions (Figure 5a). The spatial transcript dataset confirmed a correlation of LTB transcripts with

LTB-LTBR Crosstalk Is Associated with MES-like Regions of Glioblastomas
Next, we utilized a spatial transcriptomics dataset of human glioblastomas to map LTB and LTBR expression in situ [10]. We found that transcripts of LTB and its receptor LTBR are found in adjacent tumor regions with overlaps at the intercept of these regions (Figure 5a). The spatial transcript dataset confirmed a correlation of LTB transcripts with the NK exhaustion marker gene KLRG1 and lymphoid cell markers, including TRAC and IL2RA, while LTBR transcripts were associated with the pan-myeloid marker AIF1/Iba-1, the macrophage marker CD163 and the neutrophil marker CTSG (Figure 5b). Spatially weighted correlation analysis for LTB showed a strong association with MES-like glioblastoma regions with reactive gene signatures and regions showing developmental gene expression patterns, such as the OPC-and NPC-like regions (Figure 5c). LTBR expression was spatially correlated to MES-like regions with myeloid and reactive gene expression programs (Figure 5c). These findings were further confirmed by an unbiased gene ontology term enrichment analysis of differentially expressed genes in spatial dots expressing LTB and LTBR, respectively. LTB-expressing dots showed enrichment of adaptive immunity and nervous system development terms (Figure 5d). LTBR-expressing dots were characterized by the enrichment of innate immune-related gene ontology terms (Figure 5e). In situ protein validation from glioma samples of the Human Protein Atlas [57] confirmed the expression of LTBR protein in different samples in cells that had the morphological features of ramified microglia cells and foamy macrophages (Figure 5f). Its ligand LTB was undetectable in glioblastoma sections due potentially to the challenges of detecting secreted proteins in immunohistochemistry. Next, we characterized the expression of LTB and LTBR in the TCGA and CGGA datasets [58,59]. The cancer transcriptional subtype assignment [4,60] was summarized elsewhere [61]. Both datasets consisted mostly of AC-like and MES-like glioblastomas (Supplementary Figure S4a). In line with the spatial gene expression data, LTBR expression was highest in MES, followed by classical (CL) samples; LTB expression was highest in MES, followed by Proneural (PN) samples (Supplementary Figure S4b). To assess the impact of LTB expression on the clinical outcome of glioblastomas, we compared overall survival in low-and high-LTB expressing samples of both datasets with the median as the cutoff. High LTB expression appeared to be associated with a reduced overall survival (Supplementary Figure S4c), whereas LTBR expression did not reach statistical significance. In summary, we found that LTB expression was spatially correlated with the expression of NK and T cell gene expression. It was mainly found in MES-like and neuroectodermal progenitor-like regions and appeared to be associated with clinical prognosis.

Discussion
Glioblastomas are aggressive brain tumors that remain incurable. As more than 50% of a glioblastoma tumor's mass consists of myeloid cells alone [62], understanding the

Discussion
Glioblastomas are aggressive brain tumors that remain incurable. As more than 50% of a glioblastoma tumor's mass consists of myeloid cells alone [62], understanding the immune compartment of the glioblastoma microenvironment may hold the key to targeted therapies for this tumor entity. In the present study, we have conducted an integrated analysis of glioma-associated immune cells during glioma progression in humans and mice. We found that NK cells show a time-dependent accumulation during the progression of murine orthotopic GL261 gliomas. Transcriptionally, NK cells adopt a dysfunctional phenotype with a downregulation of interferon genes and stimulatory receptor proteins, including CD69 and LY49D. At the same time, these cells upregulate the LTB gene, a TNF superfamily member associated with lymph organ development. In human glioblastoma tissues, LTB expression was spatially correlated with the MES-like subtype, and neurodevelopmental gene expression signatures appeared to be associated with prognosis.
The glioblastoma immune microenvironment has been subject to extensive research in recent years [14]. While a special focus has been directed at macrophages as the most common immune cells of glioblastoma [62], lymphoid and dendritic cells have been studied extensively for targeted immune therapies, including immune checkpoint inhibition, vaccines and chimeric antigen receptor (CAR) T cell therapies [63][64][65][66][67]. As immune checkpoint inhibitors have shown moderate clinical efficacy, the molecular and cellular mechanisms of glioblastoma-associated immunosuppression are under continued investigation. Interestingly, autologous tumor-infiltrating T and NK cells were shown to regain tumor lysis activity after ex vivo stimulation, underscoring the strong immunosuppressive influence of glioblastomas [68,69]. By overcoming glioblastoma-mediated immune suppression, immunocytokines have recently shown impressive results in inducing inflammation and T cell-mediated tumor cell lysis in glioblastomas in vivo [70]. Other promising approaches include CAR NK cells [71,72]. In summary, while various therapeutic strategies are being developed, the molecular mechanisms of glioblastoma-associated immune suppression and tumor cell infiltration are gradually being understood.
Accumulating evidence points toward glioblastomas co-opting neurodevelopmental programs for tumor progression. One insidious feature of this co-option is the integration of tumor cells into existing neural cell networks that drive tumor progression [6][7][8].
The immune system adapts to the de-differentiation tendency of tumors by producing immature immunosuppressive cells [73]. In this context, LTB may serve as a molecular marker of "immune de-differentiation". Its role in lymphoid organ development and NK cell maturation is well characterized [33,34]. Interestingly, fetal lymphoid tissue inducer cells are NK cell-like cells that are also found postnatally and lack interferon-γ expression [74]. A chronically inflammatory milieu in glioblastoma drives profound changes in the immune compartment [20][21][22][23]. During chronic inflammation, LTB secretion induces the formation of tertiary lymphoid structures, the ectopic lymphoid cell aggregates [75]. NK cells are not the only cells that express LTB. It is also expressed by B and T lymphoid cells, tumor-associated macrophages and monocytes. Interestingly, a recent study highlighted an NK-like phenotype in T cells showing enhanced LTB expression [26]. Some degree of transcriptional convergence in glioblastoma has been reported within the myeloid cell compartment [20,21,23] (e.g., between microglia, CNS-associated macrophages and monocyte-derived macrophages) and even between myeloid and tumor cells [16]. These observations may support the notion of glioma-associated immune de-differentiation across different cell lineages.
The present study has outlined some similarities and crucial differences in the observed NK cell transcriptional phenotype across human and murine gliomas. In both systems, we find an accumulation of NK cells with enhanced expression of LTB and a downregulation of activation markers. In humans, enhanced LTB expression was primarily associated with MES-like tumor transcriptional subtypes that show strong transcriptional similarities with the GL261 cell line [76]. Differently, we found that human NK cells show a higher expression of the NK exhaustion marker gene KLRG1 and apoptosis-associated genes [38]. This may occur due to the overall shorter observational time frames between murine gliomas (weeks) and human glioblastomas (months). Other aspects determining interspecies differences are interindividual genetic and lifestyle variations, microbiota and therapeutic interventions in the patient cohort. In summary, we found a robust cross-species NK cell transcriptional signature associated with glioma progression.
This study has several limitations that need to be considered. The murine and human data were obtained in different laboratories, leading potentially to batch effects. As a meta-analysis, the nature of this study is observational; hence, it is lacking mechanistic corroboration. To obtain insights into the functional NK cell states beyond transcriptomics, in vitro analyses are required. Furthermore, protein validation of the main marker genes across humans and mice is advisable. We have strived to address these and other limitations by using state-of-the-art single-cell RNA sequencing data integration algorithms and analyzing these data using distinct, complementary analytical pipelines. Finally, protein validation for the human tissues was achieved using a public protein expression database.

Conclusions
Here, using human and murine glioma data, we have shown a distinct NK cell transcriptional phenotype associated with glioma progression. These cells are characterized by reduced expression of activation markers and enhanced LTB gene expression. High LTB gene expression was mainly associated with MES-like and neurodevelopmental glioblastoma subtypes. The novelty of our study is the description of a previously underappreciated NK cell state that may underlie the previously reported NK cell dysfunction [69]. Our findings contribute to the understanding of the immune component of the glioblastoma microenvironment and may help facilitate the development of immune therapies for this tumor.
Supplementary Materials: Supporting information can be downloaded at https://www.mdpi.com/ article/10.3390/cancers14194915/s1, including Figure S1: Gene ontology and regulatory element analysis of murine GL261-associated NK cells; Figure S2: Cell-cell interactions of the immune cells in the datasets suggest lymphoid-to-myeloid cell communication in lymphotoxin signaling; Figure S3: Gene ontology and regulatory element analysis of human glioblastoma-associated NK cells; Figure S4: Validation of findings using TCGA and TGGA glioblastoma datasets [77].