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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 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 use of the most predictive model is essential in transferring scientific knowledge from bench to bedside. In In vivo cancer research, immunodeficient mice models are being used, but have been found with limitations. They fall short in tumor take rates, growth kinetics, and their ability to support the growth of some human Cancer cell lines. To address these limitations, we produced an immunodeficient rat, a Sprague-Dawley Rag2/Il2rg double knockout (SRG OncoRat) rat that lacks mature B cells, T cells, and circulating NK cells. Using a variety of human cell lines (VCaP, LNCaP, 22RV1) which have poor or highly variable growth kinetics in commercially available immunodeficient mouse models, we demonstrated that the SRG rat has superior tumor take rates and growth kinetics, providing a more efficient model for drug efficacy studies. Enhanced take rates enable the use of fewer animals for studies, allows for faster timelines to drug efficacy data, and overall, may provide a better platform for reproducible drug efficacy results. In addition, the SRG rat developed larger tumor volumes, allowing for serial fine needle aspirate biopsy for PK, PD and molecular analysis in the same animal throughout the course of the study without significantly affecting normal tumor growth. The SRG OncoRat is a valuable addition to current available rodent xenograft models for evaluating novel therapies. Citation Format: Bisoye Towobola Adedeji, Noto K. Fallon, Sam Moody, Valeriya Steffey, Chris Brenzel, Jack Crawford, Tseten Yeshi Jamling, Goutham Narla. Rats support cancer studies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6133.

Pluripotent stem cells exhibit cell cycle-regulated heterogeneity for trimethylation of histone-3 on lysine-4 (H3K4me3) on developmental gene promoters containing bivalent epigenetic domains. The heterogeneity of H3K4me3 can be attributed to Cyclin-dependent kinase-2 (CDK2) phosphorylation and activation of the histone methyltransferase, MLL2 (KMT2B), during late-G1. The deposition of H3K4me3 on developmental promoters in late-G1 establishes a permissive chromatin architecture that enables signaling cues to promote differentiation from the G1 phase. These data suggest that the inhibition of MLL2 phosphorylation and activation will prevent the initiation of differentiation. Here, we describe a method to seamlessly modify a putative CDK2 phosphorylation site on MLL2 to restrict its phosphorylation and activation. Specifically, by utilizing dimeric CRISPR RNA-guided nucleases, RFNs (commercially known as the NextGEN™ CRISPR), in combination with an excision-only piggyBac™ transposase, we demonstrate how to generate a point mutation of threonine-542, a predicted site to prevent MLL2 activation. This gene editing method enables the use of both positive and negative selection, and allows for subsequent removal of the donor cassette without leaving behind any unwanted DNA sequences or modifications. This seamless "donor-excision" approach provides clear advantages over using single stranded oligo-deoxynucleotides (ssODN) as donors to create point mutations, as the use of ssODN necessitate additional mutations in the donor PAM sequence, along with extensive cloning efforts. The method described here therefore provides the highest targeting efficiency with the lowest "off-target" mutation rates possible, while removing the labor-intensive efforts associated with screening thousands of clones. In sum, this chapter describes how seamless gene editing may be utilized to examine stem cell heterogeneity of epigenetic marks, but is also widely applicable for performing precise genetic manipulations in numerous other cell types.