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Background Disruption of the phenotypic landscape via epithelial-mesenchymal transition (EMT) enables prostate cancer cells to metastasize and acquire therapeutic resistance. Our previous studies demonstrated that cabazitaxel (CBZ) (second-generation Food and Drug Administration-approved taxane chemotherapy), used for the treatment of castration-resistant prostate cancer (CRPC), causes reversal of EMT to mesenchymal-epithelial transition (MET) and reduces expression of kinesin motor protein KIFC1 (HSET). The present study examined the effect of sequencing CBZ chemotherapy mediated MET on prostate tumor redifferentiation overcoming therapeutic resistance in models of advanced prostate cancer. Methods To examine the impact of androgens on the antitumor effect of CBZ, we used human prostate cancer cell lines with different sensitivity to androgens and CBZ, in vitro, and two human prostate cancer xenograft models in vivo. Tumor-bearing male mice (with either the androgen-sensitive LNCaP or the CRPC 22Rv1 xenografts) were treated with CBZ (3 mg/kg) alone, or in combination with castration-induced androgen-deprivation therapy (ADT) for 14 days. Results Cell viability assays indicate that the presence of 5α-dihydrotestosterone (1 nM) confers resistance to CBZ in vitro. CBZ treatment in vivo induced MET in LNCaP-derived tumors as shown by increased E-cadherin and decreased N-cadherin levels. Sequencing CBZ after ADT improves tumor response in androgen-sensitive LNCaP, but not in CRPC 22Rv1 xenografts. Mechanistic dissection revealed a novel association between the androgen receptor and HSET in prostate cancer cells that is inhibited by CBZ in an androgen-dependent manner. Conclusions Our findings provide new insights into the phenotypic reprogramming of prostate cancer cells to resensitize tumors to CBZ action. This evidence is of translational significance in treatment sequencing (CBZ and ADT) towards improved therapeutic benefit in patients with lethal CRPC.

RAS family proteins are important drug targets in multiple cancers due to their central role in hypertrophic and pro-mitotic signaling. KRAS is a small GTPase that is activated in response to growth factors including HGF, TGFα, and EGF. KRAS then activates the MAP kinase (MAPK; RAF/MEK/ERK) signaling pathway, driving ERK translocation to the nucleus, and resulting in transcription of pro-mitotic and pro-growth genes. Additionally, KRAS contributes to PI3K/AKT/mTOR signaling, leading to transcriptional activation as well as hypertrophic protein synthesis via mTOR. We sought to validate several RAS mutant cell-derived tumor xenograft models in the SRG OncoRat. Cells were inoculated subcutaneously in the flank of SRG rats. To date, we have validated tumor growth using the HT-1080 fibrosarcoma NRAS mutant (Gln61Lys (c.181C>A)) cell line. We have also validated a suite of KRAS mutant lines in the SRG OncoRat, including: A-549 lung adenocarcinoma (Gly12Ser (c.34G>A)); NCI-H2122 NSCLC adenocarcinoma (Gly12Cys (c.34G>T)); MIA PaCa-2 pancreatic ductal adenocarcinoma and H-358 NSCLC adenocarcinoma (Gly12Cys (c.34G>T)); Capan-2 pancreatic ductal adenocarcinoma (Gly12Val (c.35G>T)); NCI-H441 NSCLC papillary adenocarcinoma (Gly12Val (c.35G>T)); NCI-H1734 NSCLC adenocarcinoma (Gly13Cys (c.37G>T)); and MDA-MB-231 breast adenocarcinoma (Gly13Asp (c.38G>A)). All cell lines showed robust and consistent tumor growth when injected subcutaneously into the SRG OncoRat. These models provide valuable tools for pre-clinical testing of RAS inhibition. Citation Format: R. Grace Walton, Diane Begemann, Cynthia Dunn, Valeria Steffey, Abigail Ross, Razoanul Haque, Fallon K. Noto. The SRG OncoRat supports growth of numerous RAS mutant cell lines, expanding pre-clinical RAS-inhibitor testing [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A040.

Introduction The number of approved cancer drugs as well as those in development have doubled over the last decade. With this increase comes a need for validated immunodeficient animal models that allow engraftment of tumor xenografts, an important role in the drug development process. Immunodeficient rats are suited for studies that require serial tissue sampling such as blood. Under NIH guidelines, up to 88µl of whole blood from a 125g rat can be collected per day compared to 21µl of blood from a 30g mouse. Tumor size endpoints in rats are also larger at 40mm in diameter compared to 20mm in mice, contingent on other humane endpoints. This allows researchers to follow drug response or toxicology within the same animal over more frequent time intervals. The SRG is an immunodeficient rat with deletions in the recombination activating gene 2 (Rag2) and interleukin 2 receptor gamma (Il2r-γ) that results in impaired V(D)J recombination and lymphocyte maturation respectively, ensuring T, B and NK cells deficiency. In this study, we set out to examine the levels of monocytes, another key component of the immune system response as well as for immunophenotypic differences between male and female SRGs, which have not been done in previous studies. We further evaluated the growth kinetics of 20 human tumor cell lines from 12 different cancer types in the SRG. Methods Whole blood was collected from 20 SRG rats of each gender (n=40) with 2 CD (Sprague Dawley) rats of each gender used as control group (n=4). PBMCs were isolated and analyzed by flow cytometry for CD4 and CD8 positive T cells, B cells, NK cells and monocytes. Tumor cell lines were inoculated subcutaneously into individual SRGs and tumor measurements obtained at regular intervals until study or humane endpoints. Results While levels of circulating T, B and NK cells in SRGs were reduced compared to WT CD rats (p0.05). There was also no significant difference in T, B, NK cells as well as monocyte levels between male and female SRGs (p>0.05). All 20 human tumor cell lines tested in the SRG engrafted successfully and demonstrated good growth kinetics with some tumors growing more than 10,000mm3 in size. Conclusion The immunodeficient SRG has demonstrated utility in supporting the engraftment of human tumor xenografts across 12 different cancer types. An area of interest for future studies would be on the role of monocytes on humanization. Citation Format: Koh Meng Aw Yong, Christoph Eberle, Christopher Dowdy, Grace Walton, Diane Begemann, Christopher Brenzel, Fallon Noto, Stephen Festin. The SRG immunodeficient rat demonstrates utility across multiple tumor types of different organ origins [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 51.

Human cancer xenografts are a vital tool for understanding tumor biology, growth kinetics, and therapeutic efficacy using animal models. Historically, immunodeficient mice have been the standard rodent species for cancer xenograft modeling. However, an immunodeficient rat that supports a wide variety of human cancer cell types would provide a larger rodent strain for easier surgical manipulation, serial blood sampling, and provide a single model in which efficacy, pharmacokinetics, and toxicology can be performed. We have created a Sprague Dawley Rag2 -/-, Il2rg -/- rat (SRGTM OncoRat®) that provides a highly supportive environment for growing tumors of human origin. The SRG rat lacks B, T, and NK cells and readily supports the growth of multiple human cancer cell lines. The SRG rat is more immunodeficient than the Nude rat, suggesting it may be permissive to a wider variety of human cancer types. Here we demonstrate the utility of the SRG rat for both subcutaneous and orthotopic xenograft modeling. The SRG rat supports the growth of both lung and liver orthotopic cancers. In addition, the SRG rat supports the growth of orthoptic human glioblastoma multiforme in the brain. We use in vivo imaging to visualize tumor establishment and growth in subcutaneous, orthotopic, and metastatic models. Furthermore, our data show the ability of the SRG rat to support the growth of multiple different human cancer cell types subcutaneously in two different matrices, Matrigel® and VitroGel®. These data confirm that the SRG rat is an excellent host for studying human cancer. Our data demonstrate that the SRG rat has a high utility for studies using in vivo imaging, orthotopic tumor implantation, and standard subcutaneous tumor modeling. As the most immunodeficient rat commercially available, the SRG rat supports the growth of multiple human cancer types in a larger rodent strain relative to commercially available mouse models. Citation Format: Diane Begemann, Aida Javidan, Cynthia Dunn, Nicolas Johnston, R. Grace Walton, Valeriya Steffey, Ian Corbin, Niveen Fulcher, Cleusa De Oliveira, Hu Xu, Mila Uzelac, Andrew Deweyert, John A. Ronald, Susanne Schmid, Matthew O. Hebb, Fallon K. Noto. In vivo subcutaneous and orthotopic cancer xenograft modeling in the SRG immunodeficient rat [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 42.

Human cancer xenografts are a vital tool for understanding tumor biology. While in vivo studies are traditionally done in immunocompromised mice, we have created the SRG OncoRat® that is an excellent host for human xenografts. In vivo luminescent imaging is routinely used in mouse models, and it is particularly useful for studying orthotopic xenografts or metastatic models. In this study, we validated the SRG rat as a model that can effectively be used for in vivo imaging of human cancers. For the current study, we assessed two tumor models. OV81.2-luc is a luciferase positive cell line that was generated from ascites collection from a grade IIIC serous ovarian carcinoma. Tumors were established by inoculating OV81.2-luc cells IP into female SRG rats and female NSG mice, then treated with vehicle or cisplatin for 28 days. Human non-small cell lung cancer line H358-luc is luciferase positive and metastasizes to lung when injected subcutaneously into the hind flank of SRG rats. Tumors were measured thrice weekly with calipers. For both studies, we performed weekly in vivo luciferase imaging using Spectral Instruments AMI-HT. Tumors established rapidly for both OV81.2-luc and H358-luc in both SRG rats and NSG mice, with luciferin positive signal starting one week post inoculation. In OV81.2-luc hosting animals, upon necropsy, tumors were found on multiple abdominal organs including the peritoneum, ovary, mesentery, intestines, kidneys, liver, and body cavity wall. Metastatic events outside the abdominal cavity were not evident in OV81.2-luc hosting animals. In H358-luc tumor bearing rats, a high metastatic tumor burden was found in the lungs. These data confirm that the SRG rat is an excellent host for studying human cancer when compared to commonly used immunodeficient mouse models. Data demonstrate that the SRG rat has a high utility for studies using both in vivo imaging, such as orthotopic tumor implantation, and studies on metastasis. As the most immunodeficient rat commercially available, the SRG rat retains the ability to establish human tumors while possessing size, physiology, and metabolism-based advantages when compared to mice. Citation Format: Diane Begemann, Nicolas Johnston, Marissa O’Callaghan, Grace Walton, Valeriya Steffey, Fallon Noto. In vivo imaging of ovarian and non-small cell lung cancer models hosted in the Sprague-Dawley Rag2 null Il2rgamma null SRG rat (OncoRat®) [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr A040.

Human cancer xenografts are a vital tool for understanding tumor biology, growth kinetics, and therapeutic efficacy using animal models. While in vivo studies are traditionally done in immunocompromised mice, we have created a Sprague Dawley Rag2 -/-, Il2rg -/- rat (SRG࣪ OncoRat®) that is an excellent host for human xenografts. Lacking B, T, and NK cells, the SRG rat readily supports the growth of multiple human cancer cell lines, including lines that do not engraft well or grow consistently in existing mouse models. In vivo luminescent imaging has routinely been used in mouse models, and is particularly useful for studying orthotopic xenografts or metastatic models. In this study, we validated the SRG rat as a model that can effectively be used for in vivo imaging of human cancers. For the current study, we assessed two tumor models. Ovarian cancer cell line OV81.2-luc is a luciferase positive cell line that was generated from ascites collection from a grade IIIC, serous ovarian carcinoma. Orthotopic tumors were established by inoculating OV81.2-luc cells intraperitoneally into female SRG rats and female NSG mice, which were treated with either vehicle or cisplatin chemotherapy for 28 days. Human non-small cell lung cancer line H358-luc is a luciferase positive cell line that, when injected subcutaneously into the hind flank of SRG rats, metastasizes to the lungs. Tumors were established by inoculating H358-luc cells subcutaneously into male SRG rats, and measured thrice weekly with calipers. For both studies, tumorgenicity was measured via weekly in vivo luciferase imaging using Spectral Instruments AMI HT system. Tumors established rapidly for both OV81.2-luc and H358-luc in both SRG rats and NSG mice, with visible luciferin positive signal starting one week post cell inoculation. In OV81.2-luc hosting animals, upon necropsy, tumors were found on multiple abdominal organs including the peritoneum, ovary, mesentery, intestines, kidneys, liver, and body cavity wall. Metastatic events outside the abdominal cavity were not evident in OV81.2-luc hosting animals. In H358-luc tumor bearing rats, a high metastatic tumor burden was found in the lungs. These data confirm that the SRG rat is an excellent host for studying human cancer when compared to commonly used immunodeficient mouse models. Data demonstrate that the SRG rat has a high utility for studies using both in vivo imaging, such as orthotopic tumor implantation, and studies on metastasis. As the most immunodeficient rat commercially available, the SRG rat retains the ability to establish human tumors while possessing size, physiology, and metabolism-based advantages when compared to mice. Citation Format: Diane Begemann, Fallon Noto. In vivo imaging of ovarian and non-small cell lung cancer models hosted in the Sprague-Dawley Rag2 -/-Il2rg -/- SRG rat [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2464.

BACKGROUND: Prostate cancer progression is navigated by the androgen receptor (AR) and transforming-growth factor-β (TGF-β) signaling.Wepreviously demonstrated that aberrant TGF-β signaling accelerates prostate tumor progression in a transgenic mouse model of prostate cancer via effects on epithelial-mesenchymal transition (EMT), driving castration-resistant prostate cancer (CRPC). METHODS: This study examined the antitumor effect of the combination of TGF-β receptor I (TβRI) inhibitor, galunisertib, and FDA-approved antiandrogen enzalutamide, in our pre-clinical model. Age-matched genotypically characterized DNTGFβRII male mice were treated with either galunisertib and enzalutamide, in combination or as single agents in three “mini”-trials and the effects on tumor growth, phenotypic EMT, and actin cytoskeleton were evaluated. RESULTS: Galunisertib in combination with enzalutamide significantly suppressed prostate tumor growth, by increasing apoptosis and decreasing cell proliferation of tumor cell populations compared to the inhibitor as a monotherapy (P < 0.05). The combination treatment dramatically reduced cofilin levels, actin cytoskeleton regulator, compared to single agents. Treatment with galunisertib targeted nuclear Smad4 protein (intracellular TGF-β effector), but had no effect on nuclear AR. Consequential to TGF-β inhibition there was an EMT reversion to mesenchymal-epithelial transition (MET) and re-differentiation of prostate tumors. Elevated intratumoral TGF-β1 ligand, in response to galunisertib, was blocked by enzalutamide. CONCLUSION: Our results provide novel insights into the therapeutic value of targeting TGF-β signaling to overcome resistance to enzalutamide in prostate cancer by phenotypic reprogramming of EMT towards tumor re-differentiation and cytoskeleton remodeling. This translational work is significant in sequencing TGF-β blockade and antiandrogens to optimize therapeutic response in CRPC.

Prostate cancer is a widespread problem among men, with >160 000 new cases in 2017 alone. Androgen deprivation therapy is commonly used in prostate cancer treatment to block androgens required for cancer growth, but disease relapse after androgen deprivation therapy is both common and severe. Changes in androgen receptor signaling from androgen deprivation therapy have been linked to therapeutic resistance and tumor progression. Resistant cells can become reprogrammed to undergo epithelial-mesenchymal transition, a phenotypic switch from benign, epithelial cells to a mobile cell with mesenchymal traits. In these cells, attachment to their epithelial cell layer is no longer required for survival. Anoikis is a form of cell death that occurs when detachment from other cells and the basement membrane occurs. Epithelial cells have been shown to undergo epithelial-mesenchymal transition, avoid anoikis induction and progress to a metastatic phenotype. In prostate cancer progression to advanced disease, epithelial-mesenchymal transition induction (characterized by loss of epithelial cellular attachment protein E-cadherin) correlates with a higher Gleason score, tumor progression, increased metastasis and higher biochemical recurrence. The concept of interfacing epithelial-mesenchymal transition with anoikis in the tumor microenvironment landscape will be discussed here, with focus on the significance of the functional exchange between the two processes in therapeutic targeting of advanced disease. The current evidence on the impact of loss of cell-cell contact, acquisition of chemoresistance, immune escape and metastatic spread in advanced tumors in response to transforming growth factor-β on prostate cancer metastasis will be also discussed. The signaling cross-talk between transforming growth factor-β and androgen receptor signaling will be interrogated as a new therapeutic platform for the development of combination strategies to impair prostate cancer metastasis.

Human tumor xenografts are a staple tool for understanding tumor biology, growth kinetics, and therapeutic efficacy. While these studies are most commonly done in immunocompromised mice, we have created a Sprague Dawley Rag2 null, Il2rgamma null SRGTM rat that is an excellent host for human xenografts (OncoRat®). Lacking B, T, and NK cells, the SRG rat readily supports the growth of multiple human cancer cell lines, including lines that do not engraft well or grow consistently in existing mouse models. The tumor microenvironment (TME) is a critical factor for supporting xenograft tumors, and the microenvironment of a human tumor grown in the rat has yet to be fully characterized. In this study, a collaborative effort between research institutions discovered that the tumor microenvironment in the SRG rat is more robust, involved, and more supportive of human tumor growth than in NSG mice. To characterize the aforementioned differences in rat and mouse TME, human prostate cancer cell lines LNCaP and VCaP were grown in NSG mice and SRG rats. Formalin fixed paraffin embedded sections were stained via immunohistochemistry (IHC) for both rat and mouse tumor microenvironment markers. Collagen marker CD29, endothelial cell marker CD31, macrophage marker CD45, smooth muscle actin, and stromal markers CD54 and vimentin were analyzed in both animal hosts. When applicable, staining was quantified via counting positive cells per high powered field of view. When VCaP and LNCaP xenograft tumors are hosted by SRG rats, the host TME is significantly more involved within the human tumor, and readily supports tumor growth. Comparing the same markers in SRG rat and NSG mouse hosts revealed a stark difference - the SRG rat TME is more prevalent than the mouse. Results show significantly increased stromal cells per high powered field in the SRG rat when compared to tumors of the same cell line grown in the NSG mouse. There is heavily increased endothelial and stromal cell infiltration from the host into the human tumor, and higher heterogeneity within the cell population in tumors hosted by SRG rats. These data show that there are more human tumor epithelial cell interactions within the TME of the SRG rat than in NSG mouse. Increased stromal involvement more accurately recapitulates a human TME and may help explain the better take rates and faster growth rates of xenografts in SRG rats versus NSG mice. It is well known that recapitulating the tumor cell population heterogeneity is a study limitation when using animal models. Utilization of the SRG rat TME has great value in nonclinical research by more accurately translating into human disease, while remaining in a readily available immunodeficient animal model (i.e., OncoRat). Citation Format: Diane Begemann, Bisoye Towobola Adedeji, Valeriya Steffey, Sam Moody, Goutham Narla, Fallon Noto. Sprague-Dawley Rag2 null Il2rgamma null SRG rat (OncoRat®) has enhanced tumor microenvironment in human prostate cancer xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2949.

The androgen receptor (AR) plays a critical role as a driver of castration-resistant prostate cancer (CRPC). Our previous studies demonstrated that disruption of transforming growth factor-β (TGF-β) signaling via introduction of dominant-negative transforming growth factor-β type II receptor (DNTGFβRII) in the prostate epithelium of transgenic adenocarcinoma of the prostate mice accelerated tumor. This study investigated the consequences of disrupted TGF-β signaling on prostate tumor growth under conditions of castration-induced androgen deprivation in the preclinical model of DNTGFβRII. Our results indicate that in response to androgen deprivation therapy (ADT) the proliferative index in prostate tumors from DNTGFβRII mice was higher compared with prostate tumors from TGFβRII wild-type (WT) mice, whereas there was a reduced incidence of apoptosis in tumors from DNTGFβRII. Protein and gene expression profiling revealed that tumors from DNTGFβRII mice exhibit a strong nuclear AR localization among the prostate tumor epithelial cells and increased AR messenger RNA after ADT. In contrast, TGFβRII WT mice exhibited a marked loss in nuclear AR in prostate tumor acini (20 weeks), followed by a downregulation of AR and transmembrane protease serine 2 messenger RNA. There was a significant increase in nuclear AR and activity in prostate tumors from castrate DNTGFβRII compared with TGFβRII WT mice. Consequential to aberrant TGF-β signaling, ADT enhanced expression and nuclear localization of Smad4 and β-catenin. Our findings support that under castrate conditions, aberrant TGF-β signaling leads to AR activation and β-catenin nuclear localization, an adaptation mechanism contributing to emergence of CRPC. The work defines a potentially significant new targeting platform for overcoming therapeutic resistance in CRPC.

288 Background: Epithelial-mesenchymal transition (EMT) is a significant contributor to PCa metastatic progression and therapeutic resistance in patients treated with the androgen receptor (AR) directed therapies. We previously demonstrated that aberrant TGF-β signaling accelerates prostate tumor progression in the TRAMP mouse model of tumorigenesis via selective effects on EMT. Methods: We hypothesize that the combination of the TGF-β receptor inhibitor, galunisertib (G), and enzalutamide (E) will perturb the interactive signaling between TGF-β and AR signaling affecting the phenotypic landscape of EMT. This perturbation may be exploited in our mouse model, towards enhanced anti-tumor efficacy in advanced castration-resistant PCa (CRPC). We treated 2-week old mice for two weeks with the G (75mg/kg) and/or E (30mg/kg) in combination and as single agents. Results: Treatment with G alone or in combination with E resulted in a significant reduction in prostate tumor weight without affecting total body weight. Immunohistochemical (IHC) and Western blot analysis showed that, while treatment with the G alone led to increased apoptosis and decreased cell proliferation, combination of G and E had significantly higher efficacy in inducing apoptosis and inhibiting cell proliferation than either E or G alone. As expected treatment with the G decreased the levels of nuclear Smad4 protein; the combination of G and E further decreased nuclear Smad4 expression. Furthermore the combination of G and E reversed phenotypic EMT to MET (mesenchymal-epithelial-transition), as assessed by the increase in E-cadherin among the prostate tumor cell populations. IHC and Western blot analysis also revealed that the combined treatment of G and E led to a significant decrease in nuclear AR levels compared to E-only-treated or vehicle-control tumors. Conclusions: These results provide significant insights as to the therapeutic impact of G to effectively impair the TGF-β signaling and overcome resistance of PCa patients to E by reversing EMT to potentially sensitize tumors to the antiandrogen effect. This study has major translational relevance; the combination of G and E may lead to synergistic anti-tumor impact in patients with CRPC.