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Oncology drug development has markedly lower success rate (3.4%) than other indications (20.9%) as reported by Wong, Siah and Lo in 2019. Animal models are often pointed at as the culprits for failure in clinical trials. While better animal models for cancer are needed and constantly developed, the incentive of drug developers to use those might not be clear, because the regulatory authorities do not require it. Subcutaneous tumors have earned their place in drug development but adding more sophisticated models to the testing panel before entering the clinical phase should improve the success rate. We present here the properties of subcutaneous and orthotopic tumor growth in two syngeneic models. HM-1 murine ovarian cancer cells were inoculated subcutaneously (s.c.) and intraperitoneally (representing the stage of intraperitoneal carcinomatosis) and murine renal adenocarcinoma Renca cells s.c. and orthotopically within the renal capsule. Body weight and tumor growth were followed, and mice were sacrificed according to humane endpoint criteria. To compare tumor growth in s.c. sites and within the abdomen, the tumor weight at sacrifice was divided by the number of days since the inoculation. In the HM-1 model, the mice with intraperitoneal tumor were sacrificed due to build-up of ascites on average six days earlier that the mice with s.c. tumor. In s.c. HM-1 tumor-bearing mice, the reason for sacrifice was tumor ulceration. In the Renca model, the s.c. and intrabursal models were approximately the same length. There was a striking difference in the tumor growth rate (mg/day) in s.c. versus abdominal sites in both models. The growth was 4-5 times faster in the abdominal sites than subcutaneously, highlighting the effect of tumor specific microenvironment. It is easy to understand that having promising results might be easier in a slower growing model. However, since both ovarian and renal cancer show very different growth kinetics within the abdominal cavity, testing drug candidates aimed for those indications in orthotopic models before accessing the clinical trials could give a more relevant evaluation of the potency of the drug candidates. Citation Format: Mari I. Suominen, Justyna Zdrojewska, Yvonne Konkol, Katja M. Fagerlund, Mervi Ristola, Jukka P. Rissanen. Comparison of subcutaneous and orthotopic tumor growth in syngeneic mouse models of ovarian and renal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 49.

Purpose: Radium-223 dichloride (radium-223, Xofigo), a targeted alpha therapy, is currently used for the treatment of patients with castration-resistant prostate cancer (CRPC) with bone metastases. This study examines the mode-of-action and antitumor efficacy of radium-223 in two prostate cancer xenograft models. Experimental Design: Mice bearing intratibial LNCaP or LuCaP 58 tumors were randomized into groups (n = 12–17) based on lesion grade and/or serum PSA level and administered radium-223 (300 kBq/kg) or vehicle, twice at 4-week intervals. X-rays and serum samples were obtained biweekly. Soft tissue tumors were observed macroscopically at sacrifice. Tibiae were analyzed by gamma counter, micro-CT, autoradiography and histology. Results: Radium-223 inhibited tumor-induced osteoblastic bone growth and protected normal bone architecture, leading to reduced bone volume in LNCaP and abiraterone-resistant LuCaP 58 models. Furthermore, radium-223 resulted in lower PSA values and reduced total tissue and tumor areas, indicating that treatment constrains prostate cancer growth in bone. In addition, radium-223 suppressed abnormal bone metabolic activity as evidenced by decreased number of osteoblasts and osteoclasts and reduced level of the bone formation marker PINP. Mode-of-action studies revealed that radium-223 was deposited in the intratumoral bone matrix. DNA double-strand breaks were induced in cancer cells within 24 hours after radium-223 treatment, and PSA levels were significantly lower 72 hours after treatment, providing further evidence of the antitumor effects. Conclusions: Taken together, radium-223 therapy exhibits a dual targeting mode-of-action that induces tumor cell death and suppresses tumor-induced pathologic bone formation in tumor microenvironment of osseous CRPC growth in mice. Clin Cancer Res; 23(15); 4335–46. ©2017 AACR.

Background: Chronic nonbacterial prostatitis associated with lower urinary tract symptoms (LUTS) is a prevalent condition in men. One potential pathophysiological factor is change in sex hormone, testosterone and estrogen, balance. Inflammation, cancer and obstructive voiding has been induced in the Noble rat strain by altering levels of sex hormones. We evaluated if imbalance of sex hormones could induce comparable diseases also in a less estrogen sensitive Wistar strain rats. Methods: Subcutaneous testosterone (830 µg/day) and 17β-estradiol (83 µg/day) hormone pellets were used in male Wistar and Noble strain rats to induce prostatic diseases. The rats were followed for 13 and 18 weeks. Urodynamical measurements were performed at the end of the study under anesthesia. Prostates were collected for further histological analysis. A panel of cytokines were measured from collected serum samples. Results: Noble rats exhibited stromal and glandular inflammation after 13 weeks that progressed into more severe forms after 18 weeks of hormonal treatment. CD68-positive macrophages were observed in the stromal areas and inside the inflamed acini. CD163-positive macrophages were present in the stromal compartment but absent inside inflammatory foci or prostate acini. Thirteen-week hormonal treatment in Noble rats induced obstructive voiding, which progressed to urinary retention after 18-weeks treatment. In the Wistar rats 18-week treatment was comparable to the 13-week-treated Noble rats judged by progression of prostatic inflammation, being also evident for obstructive voiding. Incidence of PIN-like lesions and carcinomas in the periurethal area in Noble rats were high (100%) but lower (57%) and with smaller lesions in Wistar rats. Serum cytokines leptin, CCL5, and VEGF concentrations showed a decrease in the hormone-treated rats compared to placebo-treated rats. Conclusions: Prostate inflammation and obstructive voiding developed also in the Wistar rats but more slowly than in Noble rats. Male non-castrated Wistar strain rats may thus be suitable to use in studies of pathophysiology and hormone-dependent prostate inflammation and obstructive voiding.

Galactoglucomannan (GGM) is the main hemicellulose class in wood of coniferous trees and could be potentially utilized as a possible health-promoting substance for food and pharmaceutical industry. Our aim was to evaluate effects of orally administered GGM-rich extract from Norway spruce in a rat model of chronic prostatitis associated with lower urinary tract symptoms (LUTS). Prostatic inflammation and LUTS was induced in male rats using testosterone and 17β-estradiol exposure for 18 weeks. Rats were treated with 2% GGM dissolved in drinking water during weeks 13-18. Pelvic pain response, LUT function and histopathological evaluation of the prostate were assessed. The results show that hormonal exposure induced LUTS seen as decreased urine flow rate, increased bladder pressure, voiding times, bladder capacity and residual urine volumes. GGM had positive effects on urodynamical parameters by decreasing the basal bladder pressure, increasing the urine flow rate and volume, reducing the residual volume and increasing micturition intervals. GGM reduced the extent of the hormone exposure-induced prostatic inflammation. Increase of pelvic pain induced by hormone exposure was only slightly affected by GGM treatment. The results suggest that orally administered GGM may have potential usage for improving lower urinary tract function associated with chronic prostatic inflammation.

Aims The aim was to study the effect of intravesically instilled cis-urocanic acid (cis-UCA) on bladder function in an experimental rat model of acute bladder inflammation. Hyaluronic acid (HA) was used as a comparator compound. Methods Bladder irritation was induced in female rats by intravesical hydrochloric acid (HCl) infusion. Vehicle, 0.5% HA, or 2% cis-UCA solutions were infused intravesically twice a day for three consequent days. On the fourth day, urodynamical measurements were performed, the animals were sacrificed, and the bladders were removed for histopathological assessment. Results HCl treatment caused significant impairment of bladder function indicated by decreased micturition intervals and voided urine volumes and induced severe voiding dysfunction observed as occurrence of overflow incontinence. These functional changes were accompanied by increased bladder weight, hemorrhage, and infiltration of inflammatory cells into the urothelium. Intravesical cis-UCA treatment recovered bladder function by significantly prolonging the micturition interval, increasing the voided volume, and reducing the occurrence of overflow incontinence. All these changes were comparable to the effects of HA. Conclusions Intravesical administration of cis-UCA was able to partially recover bladder function impaired by chemical irritation. Cis-UCA may offer a novel intravesical treatment option in some inflammatory conditions of the bladder. Neurourol. Urodynam. © 2015 Wiley Periodicals, Inc.

Objectives To investigate dietary effects on the gut microbiota composition in a rat model of nonbacterial chronic prostate inflammation (CPI). Materials and methods Nonbacterial CPI was induced in the Wistar rat strain with subcutaneous testosterone and 17β-oestradiol (E2 ) hormone pellets for 18 weeks. Rats with placebo pellets served as healthy controls. Rats with CPI were stratified into two groups, which drank either plain tap water (control group) or tap water supplemented with 2% galactoglucomannan-rich hemicellulose extract (GGM group) from Norway spruce (Picea abies) for 5 weeks. Faecal samples were collected at the end of the study, total DNA was extracted, and the bacterial composition was analysed by 16S rRNA gene sequencing. In addition, faecal samples were assayed for short-chain fatty acid (SCFA) concentrations using gas chromatography. Lipopolysaccharide-binding protein (LBP) was measured in serum samples, as an indirect indicator for bacterial lipopolysaccharide (LPS) load in blood. Results The microbial biodiversity was significantly different between the treatment groups. In the rats with CPI, there was a significant increase in gut microbial populations Rikenellaceae, Odoribacter, Clostridiaceae, Allobaculum and Peptococcaceae compared with healthy rats. Conversely, levels of Bacteroides uniformis, Lactobacillus and Lachnospiraceae were decreased in rats with CPI. SCFA butyric-, valeric- and caproic-acid concentrations were also decreased in the faecal samples of the rats with CPI. In contrast, acetic acid concentrations and serum LBP were significantly elevated in CPI rats compared with healthy ones. Amongst rats with CPI, treatment with the GGM extract significantly reduced the abundance of Odoribacter and Clostridiaceae levels, and increased the B. uniformis levels compared with CPI rats drinking tap water only. In addition, GGM significantly increased the levels of butyric acid and caproic acid, and reduced the levels of LBP in serum. Conclusions Hormone-induced nonbacterial CPI in rats is associated with specific changes in gut microbiota and secondary changes in SCFAs and LPS due to gut microbiota alteration. Our results further suggest that fermentable compounds may have a beneficial effect on CPI.

The aim was to study the effect of intravesically instilled cis-urocanic acid (cis-UCA) on bladder function in an experimental rat model of acute bladder inflammation. Hyaluronic acid (HA) was used as a comparator compound.Bladder irritation was induced in female rats by intravesical hydrochloric acid (HCl) infusion. Vehicle, 0.5% HA, or 2% cis-UCA solutions were infused intravesically twice a day for three consequent days. On the fourth day, urodynamical measurements were performed, the animals were sacrificed, and the bladders were removed for histopathological assessment.HCl treatment caused significant impairment of bladder function indicated by decreased micturition intervals and voided urine volumes and induced severe voiding dysfunction observed as occurrence of overflow incontinence. These functional changes were accompanied by increased bladder weight, hemorrhage, and infiltration of inflammatory cells into the urothelium. Intravesical cis-UCA treatment recovered bladder function by significantly prolonging the micturition interval, increasing the voided volume, and reducing the occurrence of overflow incontinence. All these changes were comparable to the effects of HA.Intravesical administration of cis-UCA was able to partially recover bladder function impaired by chemical irritation. Cis-UCA may offer a novel intravesical treatment option in some inflammatory conditions of the bladder. Neurourol. Urodynam. 35:786-791, 2016. © 2015 Wiley Periodicals, Inc.

Immunotherapy for prostate cancer has recently emerged as an attractive treatment strategy. Yet, preclinical models where relationship between inflammation, stroma, tumor cells and prostate cancer progression can be studied are limited. GEMM models of prostate cancer are scarce and in xenograft models, even when in humanized mice, the role of immune system in the initiation and in progress of the malignancy cannot be studied. As the requirement to test novel immunotherapies and especially combination treatments is increasing, a preclinical model that takes into account tumor microenvironment and immune system would be highly useful to promote development of novel therapies to combat against prostate cancer. Aim of the present study was to reveal if there is role between the immune system and development of prostate cancer, and secondly, to validate a model to be utilized later in immunotherapy development. Intact 10-12 weeks old male Noble rats were s.c. implanted with slow-releasing estradiol and testosterone pellets for 6, 13 and 18 weeks. Daily release for testosterone was 0.8 mg and for estradiol 0.08 mg. Control group animals received placebo hormone pellets without hormones. Serum samples were collected during the study to monitor hormone levels, and prostates were removed and processed for histopathological evaluation at the end of the study. Hormonal treatment caused an increase in estradiol to testosterone ratio, and the prostates were enlarged. Imbalance in hormone-milieu induced inflammation in the prostate, followed by formation of prostatic intraepithelial neoplasia (PIN)-like lesions and finally adenocarcinomas in the periurethral region. Inflammatory cells, mainly T-cells were noticed in the vicinity of PIN-like lesions. During the progression of prostate cancer, inflammatory cells disappeared from the adenocarcinoma sites. In the prostate, inflammation consisting of perivascular, stromal and periglandular T-lymphocytes and intraluminal neutrophils remained. Results of this study indicate significance of hormonal milieu, especially estrogens and androgens, in the development of inflammation and progression of prostate cancer, with a key role for tumor microenvironment. Presence of lymphocytes in the proximity of PIN-like lesions during the early phases of prostate cancer, and their disappearance later in the adenocarcinomas, indicate interaction between innate and adaptive immune system and cancer. Therefore, this preclinical prostate cancer model that combines immune system and cancer can be utilized when new immunotherapies, combination treatments and prevention possibilities against prostate cancer progression are developed. Citation Format: Mari I. Suominen, Tiina Kähkönen, Yvonne Konkol, Jenni Mäki-Jouppila, Jussi M. Halleen, Jenni Bernoulli. Preclinical efficacy model to promote immunotherapy development for prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1663. doi:10.1158/1538-7445.AM2017-1663

Mesothelin (MSLN) is a 40 kDa membrane-anchored glycoprotein, involved in mediating cell-cell adhesion, metastatic spread, promotion of cell proliferation and resistance to apoptosis. Overexpression of MSLN is most prominent in mesothelioma, ovarian, lung, triple-negative breast (TNBC) and pancreatic cancers, while in healthy tissue, MSLN is confined mainly to the mesothelial cells of the peritoneum and pericardium. Several MSLN-targeting approaches are currently being investigated, including antibody drug conjugates. We describe herein a high energy, alpha-particle emitting MSLN Targeted Thorium Conjugate (MSLN-TTC). Thorium-227 (227Th) has a half-life of 18.7 days and decays via emission of an alpha particle to radium-223 (half-life 11.4 days), a calcium-mimetic used in the treatment of CRPC [1]. The MSLN-TTC comprises an anti-mesothelin monoclonal antibody covalently linked via an amide bond to a chelator moiety possessing high affinity for thorium-227. We present data from in vitro cytotoxicity assays demonstrating selective cell killing on MSLN positive cell-lines as well as in vivo efficacy in a mouse orthotopic bone xenograft model using NCI-H226 luciferase labeled cells. Experimental procedures: MSLN-TTC was prepared in high radiochemical yields and purity. In vitro cytotoxicity experiments were performed on the mesothelin-positive cell lines Ovcar-3 (ovarian), NCI-H226 (lung mesothelioma) and mesothelin-transfected HT29 (HT29MSLN/colorectal) cells. An in vivo model was established by orthotopic intratibial inoculation of luciferase-transfected NCI-H226 cells in athymic mice. Development of bone disease was monitored by luciferase activity and the extent of bone lesions determined by x-ray imaging and microCT. Results: MSLN-TTC induced specific in vitro cytotoxicity via induction of DNA double strand breaks as determined by phosphorylated histone protein H2AX. MSLN-TTC demonstrated statistical significant in vivo potency administered as a single dose of either 250 or 500 kBq/ kg in the orthotopic bone xenograft model. Animals treated with MSLN-TTC showed a) significantly reduced disease in the bone metastatic lesions b) decreased metastatic disease in the lungs and c) significant reduction in osteolytic/ osteoblastic bone lesions as evidenced by X-Ray and microCT compared to the vehicle control group. Furthermore, no significant loss in body weight was observed during the course of the study demonstrating that the MSLN-TTC was well tolerated. The data presented support the further investigation of the MSLN-TTC in bone metastatic disease. References: 1. Henriksen, G., et al., Targeting of osseous sites with alpha-emitting 223Ra: comparison with the beta-emitter 89Sr in mice. J Nucl Med, 2003. 44(2): p. 252-9. Citation Format: Urs B. Hagemann, Else-Marie Hagelin, Katrine Wickstroem, Kristine Sponheim, Roger Smeets, Jenny Karlsson, Roger M. Bjerke, Mari I. Suominen, Yvonne Konkol, Jenni Bernoulli, Jukka Rissanen, Jussi Halleen, Liv-Ingrid Oedegaardstuen, Alan Cuthbertson. A novel high energy alpha-pharmaceutical: In vitro and in vivo potency of a mesothelin-targeted thorium-227 conjugate (TTC) in a model of bone disease. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 591.

Bone metastases are frequent and fatal outcome of advanced prostate cancer. Many of the currently used preclinical models lack typical characteristics of the heterogenic human disease. We improved existing methodology by using fresh patient-derived material in a xenograft model of bone metastasis. Clinical prostate tumor specimens were collected from robotic-assisted laparoscopic radical prostatectomy operations in Turku University Hospital (Turku, Finland). Patient-derived tumor tissue of Gleason grade 8-10 tumors (n = 5) were cut into small pieces, digested overnight, and then cell suspension was intratibially inoculated into the bone marrow cavity of nude mice (n = 8-10/patient). X-ray pictures were taken and blood samples were collected once a month. Mice were sacrificed 6 months after tumor cell inoculation, and hind limbs and lungs were collected for histological and immunohistochemical analysis. PSA was measured from serum using commercial kit. X-rays demonstrated osteosclerotic bone lesions. Immunohistochemical analysis showed that 80% of tumors in bone expressed similar characteristics compared with original tumor (androgen receptor AR, prostate specific antigen PSA, proliferation marker Ki67). However, no changes were seen in serum PSA. Lung metastases were detected in 50% of tumor-bearing mice. Interestingly, the most of lung metastases were negative for AR and PSA, which indicates that the cell population that forms metastases may be undifferentiated clone of heterogenic tumor and therefore the most aggressive. Our platform provides new tools for prostate cancer research. Citation Format: Yvonne Konkol, Maija Valta, Jenni Bernoulli, Pekka Taimen, Peter Boström, Pirkko Härkönen, Jussi Halleen, Johanna M. Tuomela. Establishment of a realistic patient-derived xenograft (PDX) model for prostate cancer bone metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 637.

Introduction Clinically, bone is a very common site of metastatic spread in many cancers. In breast, and in particular cases of advanced estrogen receptor positive (ER+) cancers, the propensity of bone involvement is 85%. Similarly in lung cancer, 30-40% of patients with advanced disease develop bone metastases, and as recent advances in lung cancer therapies improve survival, the number of patients living with bone metastases is expected to increase. At the same time there is a paucity of especially ER+ and osteoblastic animal models available for the nonclinical evaluation of new treatment strategies. We present herein the development of four mouse models of breast and lung cancer suitable for screening of new therapies. Experimental procedures Human breast cancer cell lines BT-474 and MFM-223 and non-small cell lung cancer (NSCLC) cell lines NCI-H226luc and NCI-H322 were used. BT-474 is ER+, i.e. luminal B subtype and MFM-223 is basal subtype with androgen receptor (AR) expression. H226 originates from squamous cell carcinoma and H322 from adenocarcinoma of the lung. The different cell lines were inoculated in the tibia of female nude or NOD.scid mice. Half of the BT-474 inoculated mice had a s.c. slow release 17-beta estradiol pellet implanted. The formation of bone lesions was monitored by X-ray imaging. For H226 transfected with luciferase, tumor growth was also followed by bioluminescence imaging (BLI). Finally, tumor growth and type of bone lesion, i.e. ostelytic or oestoblastic, was confirmed by histology. Results Development of bone lesions was successful in 100% and 90% of animals, with or without hormonal supplementation respectively, four weeks after inoculation of BT-474 cells. Bone lesions were detected earlier in mice with estradiol pellet and were of lytic type. In contrast, bone lesions in mice without hormonal supplementation were strongly osteoblastic. For MFM-223, bone lesions were observed 4-6 weeks after inoculation and the success rate was 60% in nude mice and 70% in NOD.scid mice. For both lung cancer cell lines, 100% of the mice developed bone lesions and were detectable already two weeks after inoculation. H226luc cells developed osteoblastic-mixed lesions and H322 cells induced lytic lesions. Very interestingly, H226luc cells also formed lung metastases in all animals, as evidenced by BLI. Some lung metastases were also found in H322 inoculated mice. Conclusions Two new osteoblastic models are added to the current scarce selection and altogether four new bone lesion models representing different subtypes of breast and lung cancer were successfully established. The different types of bone reaction in these models offer a platform for studying the underlying pathways resulting in response to treatment in osteoblastic vs. osteolytic tumor microenvironment. Citation Format: Mari I. Suominen, Urs B. Hagemann, Yvonne Konkol, Jenni Bernoulli, Katja M. Fagerlund, Roger M. Bjerke, Jenny Karlsson, Jussi M. Halleen, Alan Cuthbertson. New models of breast and lung cancer bone metastases for preclinical efficacy testing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 640.

BACKGROUND. The age-related decline of the testosterone to estradiol (T-to-E2) ratio in serum is associated with the increased prevalence of prostatic inflammation and lower urinary tract symptoms suggesting obstructive voiding. The impact of the T-to-E2 ratio on the development and reversal of non-bacterial prostatic inflammation and obstructive voiding was tested in adult Noble rats. METHODS. Adult male Noble rats (n ¼ 16) were treated with estradiol (83 mg/day) and two different doses (280 and 830 mg/day) of testosterone to cause hypoandrogenic and hyperandrogenic states with elevated estrogen. After the 13-week hormonal treatment, urodynamical measurements and electrical activity recording of the rhabdosphincter muscle were performed under anesthesia. Testosterone, estradiol, and prolactin concentrations in serum were measured and inflammatory changes in the dorsolateral prostate were classified and counted. RESULTS. Histopathological and urodynamical analyses indicated that the hypoandrogenic animals with a decreased T-to-E2 ratio (10 versus > 300 in control) developed prostatic inflammation and non-obstructive voiding. The hyperandrogenic state with decreased T-to-E2 ratio of 50 decreased the aggressiveness of the inflammation and the number of inflamed acini in the prostate and caused urethral obstruction associated with rhabdosphincter dysfunction. CONCLUSIONS. Different responses of the prostatic inflammation and voiding function to the change in T-to-E2 ratio imply that non-bacterial prostatic inflammation is not a sufficient condition for the development of obstructive voiding. The present study finds no support for the idea that age- and/or obesity-related hypoandrogenic state with a decreased ratio of T-to-E2 would cause urethral obstruction. Prostate 68: 1296–1306, 2008. # 2008 Wiley-Liss, Inc.