Targeting Bombesin Peptide Receptors for Cancer Imaging: Perspective in Prostate, Lung and Breast Cancer

One of the main reasons for focus on these particular cancer as prostate and breast cancers are the most common types cancer in men and women respectively. However small and non-small cell lung cancer are most frequent in both. The molecular basis for the utility of radiopeptides is due to their peptide receptor over expression by certain tumours [1]. Peptides are more important elements in fundamental biological processes due to their specificity for controlling various biological functions through their specific receptors naturally. The increased attention for peptides or their receptors for imaging cancer is due to their expression/over expression in many primary human cancers and their ability to target specific receptor. The clinical impact of radiolabelled peptides has been observed in theranostics by diagnosis of location of tumours and their metastases by receptor scintigraphy and by facilitating receptor radiotherapy of tumours for treatment. However, the research on peptide based radioligand seems to be slight aperture in the huge oncological area, but the receptor targeting by short peptide of over expressed receptors in tumours has become very important for cancer imaging [2].


Introduction
One of the main reasons for focus on these particular cancer as prostate and breast cancers are the most common types cancer in men and women respectively. However small and non-small cell lung cancer are most frequent in both. The molecular basis for the utility of radiopeptides is due to their peptide receptor over expression by certain tumours [1]. Peptides are more important elements in fundamental biological processes due to their specificity for controlling various biological functions through their specific receptors naturally. The increased attention for peptides or their receptors for imaging cancer is due to their expression/over expression in many primary human cancers and their ability to target specific receptor. The clinical impact of radiolabelled peptides has been observed in theranostics by diagnosis of location of tumours and their metastases by receptor scintigraphy and by facilitating receptor radiotherapy of tumours for treatment. However, the research on peptide based radioligand seems to be slight aperture in the huge oncological area, but the receptor targeting by short peptide of over expressed receptors in tumours has become very important for cancer imaging [2].
The most common radio peptide analogues are the somatostatin derivative and some of these derivatives have become commercially available, FDA approved drugs [2,3]. Some of them such as, 111 In-DTPA-OctreoScanP®P, which binds to somatostatin receptor for the diagnosis of neuroendocrine tumours. 99m Tc-analogue is known as NeoTect which expressed better specificity for lung cancer diagnosis. Octreotide and its analogues labelled with 111 In,

Bombesin Receptors
A vital tetradecapeptide, Bombesin (Bn) was sequestered from the skin of two European marine toads which belongs to the Bombina toridae, Bombina bombina and Bombina variegate family. The rare name of this peptide family was originated from the names of the frogs or toads. However, the authentic novel amidated tetradecapeptide was isolated from the European frog Bombina bombina [9,10]. Subsequently many similar peptides were reported and divided into three groups as per their modification in the particular amino acids (Table 1). Table 1: Bombesin and related peptide groups.
Gastrin-releasing peptide receptor (GRPR) is a BB2 subtype and the mammalian ligand for this receptor is Gastrin-releasing peptide (GRP) [12]. McDonald et al. [17] in 1979 found that GRP is 27 amino acids peptide and having similarity with C-terminal amino acid with bombesin. GRP also work as an endocrine cancer cell-growth factor for both normal tissues and cancer cells and boosts many physiological actions like exocrine secretion and smooth muscle contraction. BB2 receptors have shown their overexpression in various human cancers i.e. breast, prostate, lung, central nervous system especially gliomas & meningiomas, ovarian, pancreatic cancers and neuroblastomas [6,18,19]. The affinity of BB1 towards NMB is seen to be 100-fold higher than towards GRP. On the other hand, the affinity of BB2 receptor towards GRP is found to be 50fold higher than towards NMB [20,21].
A subtype of G protein coupled receptor having similar structure with bombesin-receptor (BnR) subtype is human bombesinreceptor subtype-3 (BRS-3). BRS-3 is having 51% amino acid similarity of the GRPR and its 47% of the amino-acid is identical with the NMBR [12,22] [12] enlightened that the BRS-3 have a restricted distribution than the GRP and NMB receptors. However, the BRS-3 is particularly expressed in GI tract along with CNS and peripheral tissues. BRS-3, NMB-R, and GRP-R have shown expression in a various human tumors and cancer cell lines at different grades [8,26]. NMB is of great interest as expressed by a various type of cancers due to their capacity to generate tumour cell proliferation [27,28]. However, GRPR subtype is more important due to its higher expression density as compare to NMBR and BRS-3. In terms of peptide, being a small regulatory peptide Bn has high cellular permeability and bio compatibility because of which it is seen to a better targeting molecule. Moreover, Bn can also be integrated with nanoparticles to increase surface density (peptides per nanoparticle) due to its nano size and does not influence the properties of the nanoconjugation [1,29].
In the first human trial of 68 Ga-BAY 86-7548 that includes 5 volunteers, the excretory route was found to be urinary system (absorbed dose = 0.61mSv/MBq), the second organ absorbing highest dose was pancreas (absorbed dose = 0.51mSv/MBq and overall effective dose was 0.051mSv/MBq [33]. Similarly, two other radiopharmaceuticals: 68 Ga-NOTA-RM26 and CT of 68 Ga-BAY 86-7548 before surgery in PCa indicates PET accuracy of 83% with almost similar or higher sensitivity (89%) and specificity (81%). The highest uptake was reported in PCa tumour site when compared with normal and benign prostate hyperplasia. Similar results were lately reported by Touijer et al. [37] who also concluded that no connection was observed in GRPR and prostate-specific membrane antigen (PSMA) expression using immunohistochemistry.
The role of 18 F and 64 Cu tagged antagonist were also assessed in a small study of PCa patients. In a study of 4 patients, 64 Cu labelled antagonist ( 64 Cu -CB-TE2A-AR06) role was evaluated in recently diagnosed cases of PCa [38]; Labelling GRPR antagonists with 64 Cu not only had high tumour to background ratio but also had high image contrast, longer half-life which is important parameter for dosimetry calculations. In a comparative study of 18 F-BAY 864367 with 18 F-fluorocholine, the usefulness of 18 F-BAY derivative in human study of 10 patients with biopsy confirmed PCa were evaluated. The PET/CT of 18 F-BAY 864367 showed positive tumour lesion in 5 out of 10 patients. However, the combination of both along with histopathology experiment accurately indicates prostate lesions for 5 negative scans [39]. In breast and prostate cancer with advanced disseminated disease positive scan was depicted in only about 50% patients using radiolabelled GRPR antagonist SB3 (DOTA-paminomethylaniline-diglycolic acid-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHEt) [40]. 68 Ga-RM2 ((DOTA-4-amino-1-carboxymethyl-piperidine-D-Phe-GlnTrp-Ala-Val-Gly-His-Sta-Leu-NH 2 ) and 68 Ga-PSMA PET was comparatively studied by Minamimoto et al. [41]. They evaluated the physiological and abnormal uptake of 68 Ga-RM2. The clearance route of 68 Ga-RM2 was found to be kidneys with the highest uptake in pancreas while 68 Ga-PSMA-11 had urinary excretory route, with highest salivary uptake alongwith GI and hepatobiliary uptake. Nock et al. [42] reported the biologic profile of modified 67 Ga-, 177 Lu-, and 111 In-NeoBOMB1 based radioligands, in-vitro studies by GRPRpositive cells and in-vivo by mouse models. All three radioligand indicate high binding affinity (IC 50 =1-2nM) and good metabolic stability in peripheral mouse blood (>95% intact at 5 min after injection). High GRPR-specific uptake in the PC-3 xenografts after injection was also observed.
In a review study of 18F-fluoroethylcholine PET/CT with 16 repeated prostate cancer patients had given negative and inconclusive results. Wieser et al. [43] compared it with 68 Ga-RM2. PET/CT of 68 Ga-RM2 exhibited uncommon uptake in 10 patients/16 patients. In the two patients multiple bone lesions and additional lymph nodes in the pelvis was noted with indecisive 18 Ffluoroethylcholine PET/CT. The raised PSA levels during the scan of 68 Ga RM2 PET/CT (5.5ng/ml) then at 18F-fluoroethylcholine PET/CT (2.4ng/ml) requires confirmation with large cohort of patients. Minamimoto et al. [44] enrolled patients with biochemical relapse and negative CT, MRI and 99 Tc-MDP scan. Out of 32 patients enrolled in the study, 23 patients were identified as recurrent prostate cancer by 68 Ga-RM2. The detection rate was found to be 71.8% which indicates the potential of 68 Ga-RM2 in biochemical relapse of prostate cancer.

Zhang et al. [35] included 17 newly diagnosed patients and 11
post therapy patients of prostate cancer in the study. 68 Ga-RM26 PET/CT detected all 11 patients with biochemical recurrence and 15/17 newly diagnosed patients were also positive in 68 Ga-RM26 PET/CT. 68 Ga-bombesin PET/CT, (GRPR agonist) were performed on 22 patients, but it detected less primary tumours, lymph nodes, and bone metastases than 68 Ga-RM26 (GRPR antagonist).
A prospective study in 95 patient having biochemical recurrent prostate cancer was treated with 68 Ga-RM2 PET/MRI observed the highest uptake in urinary bladder, which indicate the complex was excreted through urinary route [45]. Pancreas was the organ with the highest uptake followed by intense uptake in GI tract. Mild to moderate uptake was also observed in stomach, duodenum and rectum. In another study (15 patients) of biopsy confirmed prostate cancer, correlation of PET scan before surgery and histopathology were presented. They found no correlation in region based and whole prostate SUV max with histopathology and postoperative T category [46].

Theragnostic studies
Kurth et al. [47] performed first human dosimetry of 177 Lulabelled GRPR antagonist in 4 patients (mean activity=4.48±0.9GBq) with metastatic castration-resilient prostate cancer. According to RADAR dosimetry scheme and based on quantitative SPECT/CT, absorbed doses for different organs along with tumour lesions were considered. The mean absorbed dose was highest in pancreas (1.08±0.44Gy/GBq) as suggested by diagnostic studies. Tumour lesion received mean dose of 6.20±3.00Gy/GBq [47]. Similar to PSMA ligand studies, the bone marrow shows low uptake with 177 Lu-RM2 [48]. In case of kidneys, 177 Lu-RM2 showed a low absorbed dose as compared to that of 177 Lu-PSMA-617 [48] or 177 Lu-DOTA-TATE [49].
GRPR-targeted radiopharmaceuticals are effective radiotracers for prostate cancer assessment, indicating a higher rate of detection; they are also reliable for metastatic foci evaluation. These initial reports are encouraging but further study is required to find out the association of GRPR & advanced hormone-resistant prostatic tumours expression.

Copyright © Raunak Varshney
NACS.000611. 5(3).2020 [12,8,51]. There is increased, stimulus for growth and proliferation as well as increased adhesion when Bombesin receptors were activated on lung cancer subtypes [28, 50,52]. The higher expression of GRPR is mostly observed in the patients having advanced stage of lung cancer [51]. However, limited study to image/target lung cancer cells over expressing bombesin receptors is available. The use of BnR-labelled probes to diagnose lung-cancers in humans is still under research. In another study 99m Tc radiolabelled multimeric receptor targeting probe using RGD tripeptide to target α v β 3 -integrins and bombesin receptor-agonist was used. The probe indicated higher uptake for the possibility to image the lung cancer cell metastasis in-vivo by targeting multiple receptors [53]. In another study 99m Tc-labelled-GRPR-agonist imaging resulted in high tumour to background ratio in lung cancer xenograft nude mice model, suggesting this might be good imaging methodology for NSCLC [54].
Mattei et al. [51] stated that in case of advanced-stage lung disease the expression GRP receptors was found to be more prevalent and a strong correlation between the stage of lung cancer and GRPR expression was observed. Comparison of GRPR expression in a small cell and non-small cell lung cancer shows almost similar results; however, the GRP-receptor expression has increased intensity in non-small cell lung cancer.
BRS-3-receptor is also known as bombesin receptor due to their structural homology. It is due to the fact that several studies have mentioned in lung cancer cells having growth properties in these cells. Moreno et al. [55] confirmed the prospective significance of orphan BRS-3 receptor. They have examined the incidence/comparative assessable manifestation of human BRS-3 and GRPR/NMBR receptor along with its effects for initiation on cell-signalling/progress in various human lung-cancer cell-lines.
The findings indicate that human BRS-3, much like GRPR/NMBR, is frequently present at high levels (62%) in lung cancer cells. These results indicate that BR-3 must be studied in detail like GRPR/ NMBR for the development of new treatment, diagnosis or both for lung cancer.

Bombesin receptors and breast cancers
Numerous studies show that in breast cancers, 38-96% is of GRPR and 0-50% is of NMBR, BRS-3 subtypes [8,25,56,57]. Many authors have extensively demonstrated overexpression of GRP receptors in breast cancer [8,[56][57][58][59], explicitly in estrogen receptor expressing class cancers [60][61][62]. Many radiolabelled SPECT ( 111 In, 99 mTc) and PET ( 64 Cu, 68 Ga, 18 F) BnR-agonists are predominately used for GRPR imaging. These agents bind to breast-cancer-cells and indicated good result both in cell line and animal models. Few authors have also carried out a comparative in vivo study using 68 Ga-labelled-BnR-agonist and common PET-imaging agents on xenografts of breast cancer in nude-mice. The results indicated higher tumour-uptake in case of 18 F-FDG and 68 Ga-labelled-BnRagonist than 18 F-FDG alone. Moreover, 68 Ga-labelled-BnR-agonist showed tumoral changes as a result of tamoxifen treatment which were not obtained using 18 F-FDG. It was concluded that along with better tumour imaging, 68 Ga-labelled-BnR-agonist also helped in hormonal-treatment response assessment in breast-cancer-cells which was not possible using 18 F-FDG [63][64][65].
Liu Z et al. [66] developed heterodimeric-PET-probes based on 18 F, 64 Cu and 68 Ga radioisotope by conjugating the RGD sequence to target α v β 3 -integrins and BnR-agonist. High-uptake of agonist and capability to image in xenografed nude-mice was observed. This conjugate permit to image breast-cancers not only with high expressing Bn receptor-but also low expressing α v β 3 -integrinreccptors due to dual receptor targeting. Iron-oxide nanoparticles conjugated BnR agonists were evaluated by Jafari et al. [67] and this specific contrast agent has high affinity for GRP receptor on breast cancer cell lines for MR-Imaging. Additionally, it has shown good diagnostic potential to localize breast cancer-in-vivo. Aranda Lara L et al. [68] evaluated a fused probe integrating a 99m Tc-BnR agonist and folate-receptor to target constantly overexpressed receptors GRPR and folate (FRα) in breast cancer. It showed increased uptake both in in vitro and in vivo studies using breastcancer-cell line and breast-cancer-xenografts respectively. Heidari et al. [69] investigated the use of BnR-agonists conjugated to gold-nanoparticles/nano rods coated with polyethylene glycol as a potential nonionizing laser based-imaging-agent having higher affinity for GRPR expressing breast cancer cells. De Barros assessed 99m -Tc labelled-BnR-agonist conjugated with liposome which exhibited high-uptake and strong scintigraphic images of breastcancers [70,71]. Furthermore, Guojun W et al. [72] revealed that the development of MDA-MB-231-breast cancer cells was inhibited by a NMBR antagonist.
There are many clinical studies using radiolabelled BnR agonist probes in detecting, localizing and imaging breast lesions [73][74][75][76][77][78][79][80][81]. In a study by Van de Wiele C et al. [73] high specific tumourlocalization and better tumour imaging were observed in 4/6 breast tumour patients using radiolabelled-BnR-agonist-ligand ( 99m Tc-RP527). In other study they have evaluated nine clinically suspected breast cancer and four 4-tamoxifen-resistant osseous malignancies from breast-cancers [74]. In which the primary tumour uptake of 99m Tc-RP527 was evident in 8 patients in a group of 9 patients and in case of tamoxifen-resistant patients no uptake of 99m Tc-RP527 was found.
A comparative study of 99m Tc-Bn and 99m Tc-alone in 3 breast cancer patients was reported by Scopinaro F et al. [75]. They have mentioned higher absorption in 99m Tc-labelled-Bn probe in the tumour than 99m Tc. The lymph node metastases were also detected by 99m Tc-labeled-Bn demonstrating potential use of this technique. Four breast cancer patients and seven healthy subjects were studied by Mendoza-Sanchez et al. [76] in the normal physiologic distribution of radiolabelled-BnR-agonist ( 99m Tc-HYNIC-Lys3-Bn). The maximum uptake was observed in malignant tumour followed by lungs, pancreas and renal route of excretion; however, the least uptake is observed in bone marrow. In a consecutive study on 33 Tc-Bn imaging indicates100% sensitivity, 60 % specificity and 100% negative predictive value. Similarly, 99m Tc-RGD-Bn and 99m Tc-3P4-RGD2 was reported for detection of breast cancer lesions in 6 female patients by Chen et al. [78]. The results indicate five malignant lesions with distinct uptake and in one case uptake was not distinctive. The four malignant cases have shown integrin and GRPR expression, one case indicated GRPR positive (integrin α v β 3 = -ve) while one case was integrin (α v β 3 ) positive (GRPR negative).
Ji T et al. [79] compared the results of ultrasound and 99m Tc-RGD-Bn to locate the tumour. Due to the dual receptor targeting sensitivity of 99m Tc-RGD-Bn SPECT/CT was observed 93.5% and 82% than ultrasound respectively. A similar specificity (79%) and positive predictive value was obtained for 99m Tc-RGD-Bn SPECT/ CT as well as for ultrasound. In case of lymph node metastases, the 99m Tc-RGD-Bn SPECT/CT was found to be more sensitive in detecting lesions greater than 10mm. However, with increased sensitivity 99m Tc-RGD-BBN has ability for identifying breast lesions but still its role is complementary to ultrasound.
Stoykow et al. [80] conducted a study to investigate the use of 68 Ga-RM2 in fifteen diagnosed breast cancer patients. 68 Ga-complex clearly identified thirteen lesions out of total eighteen along with five false-negative results.ER and PR expression was exhibited by all cancers as seen on PET. Among the 5 cancers only one came out to be ER-positive and other not diagnosed by PET. 68 Ga-NOTA-RM26 PET/CT was studied by Zang et al. [81] and detected 29 primary tumours out of 34 tumours which were also validated histopathologically. The uptake value of five PET-negative tumours was less than or equivalent to the uptake value of normal breast tissue. Histopathological analysis proved the occurrence of lymph node metastases in eighteen, while PET was affirmative in fifteen patients. There was significant increase in the SUVmax in ERpositive tumours comparatively to negative tumours, in addition a positive correlation was observed with GRPR expression level. It was also found that the SUVmax were considerably greater in the secretory stage as compared to non-secretory stage or the postmenopausal stage. The sensitivity and specificity of 68Ga-complex appeared to increase in the patients showing ER-negative tumours, however the patients undertook the acquisition on their secretory stage were excluded from this study.
Morgat et al. [73] comparatively evaluated 68Ga-RM2 and 18F-FDG for precise uptake in tumour areas of breast cancer (14 samples) by using tissue micro imaging. They have also examined Ki-67, HER2, GRPR and immunohistochemistry analysis for ER and progesterone receptor (PR) in all the specimens. The authors observed higher specific binding of 68 Ga-RM2 and lower of 18 F-FDG uptake in the ER-and PR-+ve type of cancer groups. However, the low-Ki-67 group of gallium derivative and high-Ki-67 group of 18 F-FDG has shown negligible difference in the prominence of HER2.They have observed that 68 GaRM2 gives higher binding in tumours independently indicating this gallium PET ligand could be corresponding to 18 F-FDG in ER-positive bearing tumours with a small proliferation manifestation. For an overview of all bombesin analogues clinical studies see (Table-2). Ga-NOTA-RM26(D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH 2 ) PET/CT Positive correlation was found between radiolabelled complex 2018 [81] In conclusion a strong correlation was observed in expression of GRPR in breast cancer cells showing ER-positive tumours. This shows high potential for development of GRPR antagonist for both diagnostic and therapeutic usage.

Conclusion
Prostate, Breast and Lung Cancers are the frequent cause of death worldwide. It is important to identify innovative and successful therapeutic methods for both early diagnosis and treatment. The over expression of various bombesin receptors in these cancers directs to advanced novel approaches for imaging as well as selective delivery of therapeutic agents. We have deliberated the role of bombesin peptide receptors in cancer imaging that could offer novel pathways to cancer diagnosis and treatment as widespread occurrence of BnR over expression in the mentioned cancer type. Several preclinical studies in animal models and recent human studies (especially breast / prostate cancer) the over-expression of these bombesin receptors showed promising results for theranostic. However, to better understand the bombesin receptors expression and to be considered as the potential perspective for diagnosis and therapy, large prospective clinical trials will be required.