Evolution Of The Rat Model

Jack Crawford on September 30, 2022

Rats have been favored for drug development studies because the metabolism and pharmacokinetic properties of drugs in rats are most similar to humans. Rats are also preferable for xenograft studies because they allow for tumor volumes 10-fold higher than in mice, they are easier to surgically manipulate, and they can accommodate multiple blood samplings to assess the pharmacokinetic properties of a drug. To successfully generate cancer xenografts from humans in rats, the animal must be immunodeficient to prevent rejection of the xenograft by the animal’s immune system.

The first generation of immunodeficient rats is the nude rat. Nude rats are characterized as being devoid of T-cells, but still retaining functional B- and NK-cells. The nude rat accepts human xenografts, but studies have shown that nude rats have increased incidences of tumor regression likely related to age-dependent changes in immunocompetence 1-2.

The answer to some of these deficiencies was the development of severe combined immunodeficiency (SCID) rats. SCID rats are Prkdc deficient which means the rat has no B- or T-cells. SCID rats demonstrate severe immunodeficiencies without the “leaky” phenotype that is observed in SCID mice – where detectable levels of Ig are generated by a few clones of functional B-cells3. SCID rats also demonstrate growth retardation and exhibit premature senescence. SCID rats host xenografts successfully, but only survive for around a year if kept under very strict pathogen-free conditions.

In order to overcome the limitations of the SCID rat, Hera Biolabs developed the Sprague-Dawley Rag2 null (SDR) rat. SDR rats are generated on a Sprague-Dawley background – an albino, outbred lab rat that is preferred for metabolism and toxicity studies. Sprague-Dawley rats are also preferred for their calm demeanor, ease of handling, and larger size than Wistar rats. The SDR rat is null for the Rag2 gene which results in a lack of B-cells and a severely reduced T-cell population4. These rats are highly permissible to xenografts and xenografts demonstrate greater uniformity in growth profiles. SDR rats have also been shown to successfully host large, rapidly developing xenografts of human cancer cell lines (e.g. H358, VCaP) that are difficult or impossible to generate in NSG mouse models. The SDR rat maintains a population of NK-cells which makes this model unlikely to accept xenografts of all tumor types.

The researchers at Hera Biolabs noted that a limitation of the SDR model is the large population of NK-cells that is maintained. To overcome this limitation, an evolution on the SDR rat has been developed at Hera biolabs that contains an additional Il2gamma null, known as the OncoRat®-SRG™. The OncoRat is completely depleted of B-cells, T-cells, and NK-cells. The OncoRat boasts an engraftment take rate of 90%+ using non-small cell lung cancer (NSCLC) patient derived xenograft model establishment as the example.

Hera - Blog - Evolution of the rat model - Figure 1

References

  1. Colston, M. J.; Fieldsteel, A. H.; Dawson, P. J., Growth and regression of human tumor cell lines in congenitally athymic (rnu/rnu) rats. Journal of the National Cancer Institute 1981, 66 (5), 843-8.
  2. Maruo, K.; Ueyama, Y.; Kuwahara, Y.; Hioki, K.; Saito, M.; Nomura, T.; Tamaoki, N., Human tumour xenografts in athymic rats and their age dependence. British Journal of Cancer 1982, 45 (5), 786-789.
  3. Mashimo, T.; Takizawa, A.; Kobayashi, J.; Kunihiro, Y.; Yoshimi, K.; Ishida, S.; Tanabe, K.; Yanagi, A.; Tachibana, A.; Hirose, J.; Yomoda, J.-i.; Morimoto, S.; Kuramoto, T.; Voigt, B.; Watanabe, T.; Hiai, H.; Tateno, C.; Komatsu, K.; Serikawa, T., Generation and Characterization of Severe Combined Immunodeficiency Rats. Cell Reports 2012, 2 (3), 685-694.
  4. Noto, F. K.; Adjan Steffey, V.; Tong, M.; Ravichandran, K.; Zhang, W.; Arey, A.; McClain, C. B.; Ostertag, E.; Mazhar, S.; Sangodkar, J.; Difeo, A.; Crawford, J.; Narla, G.; Jamling, T. Y., Sprague Dawley Rag2 null rats created from engineered spermatogonial stem cells are immunodeficient and permissive to human xenografts. Mol Cancer Ther 2018.

Syngeneic Orthotopic Brain Cancer Xenografts In Rats

Hera Admin on June 13, 2022

C6, 9L, and F98 are the three of the most used rat glioma models in research. In their recent review article, Sahu et al. describe the origin, genomics, and use of these three models in research1. To this day, gliomas remain incredibly hard to treat and patients commonly have very low survival rates. Orthotopic rat brain cancer models are central to generating better clinical interventions. Leveraging our expertise in genetically engineered cell-lines, rat surgeries, and in vivo bioluminescent imaging capabilities, Hera BioLabs is expanding our orthotopic brain offerings in rats.

Some of the advantages of rat versus mouse brain tumor models are as follows:

  • A larger brain allows more precise stereotactic implantation and a longer time until tumor endpoint
  • The larger tumor size creates better in vivo imaging
  • Therapeutic agents can be given intracerebrally (i.c.) with greater ease
  • More literature and research exist on rat brain tumors compared to their mouse counterparts

Hera BioLabs’s SRG Rat™

We offer another significant advantage – a highly immunocompromised rat model, the SRG. Lack of a complete immune system can counteract the inability to generate orthotopic data as two of these most widely used glioma models, C6 and 9L are highly immunogenic in many rat strains. If an intact immune system is not required, the SRG rat facilitates studies using immunogenic cell lines.

The C6 cell line is the most used rodent brain tumor model and expresses genes resembling those reported in human gliomas. Several of these genes are responsible for stimulation of the Ras pathway. C6 is widely used to evaluate therapeutic efficacy in vitro, yet it is immunogenic even in its host Wistar rat and currently has no host it can be successfully propagated in.

9L is the second most commonly used rat brain tumor model. 9L is a gliosarcoma derived from Fischer rats and develops rapidly growing tumors when implanted i.c. This cell line is primarily used to study drug transport in the brain and tumor blood barriers, drug resistance, modeling brainstem tumors, and MRI, PET, and PK studies. Similar to C6, 9L can provoke a strong antitumorigenic immune response.

F98 glioma is the third most common rat brain tumor model. Genes that are overexpressed in F98 include Ras, PDGFβ, cyclin D1/D2, EGFR, and Rb. This model simulates human GBMs closer than other cell lines due to its high invasiveness and weak immunogenicity, making it an ideal candidate for studying efficacy of therapeutic agents. F98 also has several gene-modified versions including F98-EGFR, F98npEGFRvlll, and a luciferase expressing line which allows for in-vivo imaging.

 

Hera BioLabs - Blog - Hera BioLabs Is Expanding Our Orthotopic Brain Offerings In Rats-Table 1 Image

Table 1: Comparison of C6, 9L, and F98 significant molecular markers.

 

Hera BioLabs - Blog - Hera BioLabs Is Expanding Our Orthotopic Brain Offerings In Rats-Figure 1 Image

Figure 1: Histopathological view of the C6, 9L, and F98 rat brain tumors. A. C6 glioma is composed of pleomorphic cells ranging from round to oblong nuclei with a mild herring pattern of growth and focal invasion of contiguous normal brain. B. 9L gliosarcoma is composed of spindle-shaped cells with a sarcomatoid appearance. A whorled pattern of growth is seen, with little invasion of contiguous normal brain. C. F98 glioma is composed of a mixed population of spindle-shaped cells with fusiform nuclei, a frequent whorled pattern of growth is seen with a smaller subpopulation of polygonal cells with round to oval nuclei. Extensive invasion of the contiguous brain occurs with islands of tumor cells and a central area of necrosis.

 

Work With Hera BioLabs

Reach out to us to learn more about using our SRG Rat for your research.

References

  1.  Sahu, U., Barth, R. F., Otani, Y., McCormack, R. & Kaur, B. Rat and Mouse Brain Tumor Models for Experimental Neuro-Oncology Research. J Neuropathol Exp Neurol 81, 312-329, doi:10.1093/jnen/nlac021 (2022).

Acute Myeloid Leukemia MOLM13 And Optimus

Hera Admin on May 4, 2022

The American Cancer Society estimates that approximately 60,650 new leukemia cases and 24,000 leukemia deaths will occur in 2022. Of these, acute myeloid leukemia (AML) will account for around 20,050 new cases and 11,540 deaths. Almost all will be in adults, with increased risk associated with age and male sex1. AML, the most common leukemia in adults, involves uncontrolled proliferation of myeloid stem or progenitor cells in hematopoietic tissue. These aberrant myeloid cells accumulate in the bone marrow, leading to decreased hematopoiesis, thrombocytopenia and anemia2.

Left untreated, AML progresses rapidly to death. Thus, it is imperative that pharmacological interventions work rapidly. Due to this constraint, it is believed that AML patients have often received higher than needed drug doses. Recently, the US FDA released Project Optimus, a set of guidelines aimed at refining dose optimization in the clinical setting3. The guideline has already impacted clinical pipelines for AML therapy; Kura has announced a dose-optimization trial for their AML drug KO-539 and Amgen will soon do the to do the same with their KRAS inhibitor Lumakras4.

Hera BioLabs - Blog - Acute Myeloid Leukemia MOLM13 and Optimus - Figure 1 Image

Figure 1. Bracketing Studies use more dose level groups in order to provide more information about the relationships between efficacy, safety, toxicity, and dose.

The FDA also recommends that drug developers consider Project Optimus when designing pre-clinical studies. The FDA would like to see more dose levels tested in animals, with an emphasis on assessing tumor drug exposure and performing bracketing analyses to determine optimal dosing (Figure 1).

The SRG OncoRat offers numerous advantages for performing pre-clinical AML studies in keeping with Project Optimus recommendations. Due to its larger size, the SRG rat allows for multiple types of data to be obtained from the same animals within efficacy studies, including:

  • Serial blood draws for Pharmacokinetics & pharmacodynamics
  • Serial tumor biopsies for tracking tumor drug exposure over time
  • Better take rate with difficult tumor models, such as AML
  • Lower variance in tumor growth kinetics, allowing for fewer animals and more doses

We have recently developed a solid tumor AML model in the SRG rat, allowing for maximum use of each animal. The MOLM-13 cell line was established from the blood of a young Japanese man with relapsing acute myeloid leukemia that progressed from myelodysplastic syndrome. These cells express monocyte-specific esterase, MLL-AF9 fusion mRNA, trisomy 8, as well as other trisomies that are present in various subclones5. MOLM-13 tumors exhibit rapid growth, and they are responsive to Sponsor test articles.

Hera BioLabs - Blog - Acute Myeloid Leukemia MOLM13 and Optimus - Figure 2 Image

Figure 2. MOLM-13 tumor growth. SRG rats were inoculated subcutaneously with 75,000 MOLM-13 cells and tumor growth was monitored with electronic calipers. N = 9.

The SRG OncoRat is a powerful tool for in vivo oncology studies, as it outperforms mouse models for compliance with the goals of Project Optimus. SRG rats are extremely well-suited for hosting solid AML tumors for analyses, including drug efficacy, tumor growth kinetics, and PK/PD studies. Tumor volumes are far larger than those obtained in mice, allowing for serial tumor biopsies to assess drug exposure.

If your preclinical studies could use a boost, or you would like to see more data on the SRG Rat, contact Hera BioLabs for more information here.

References

  1. Cancer Facts & Figures 2022, https://www.cancer.org/cancer/acute-myeloid-leukemia/about/key-statistics.html#references (2022).
  2. Almosailleakh, M. & Schwaller, J. Murine Models of Acute Myeloid Leukaemia. Int J Mol Sci 20, doi:10.3390/ijms20020453 (2019).
  3. Project Optimus: Reforming the dose optimization and dose selection paradigm in oncology, https://www.fda.gov/about-fda/oncology-center-excellence/project-optimus (2022).
  4. Armstrong, A. FDA’s renewed focus on oncology dosing spooks investors, but companies say they’re ready, https://www.fiercebiotech.com/biotech/fda-s-renewed-focus-oncology-dosing-spooks-investors-but-companies-say-they-were-ready (2021).
  5. Matsuo, Y. et al. Two acute monocytic leukemia (AML-M5a) cell lines (MOLM-13 and MOLM-14) with interclonal phenotypic heterogeneity showing MLL-AF9 fusion resulting from an occult chromosome insertion, ins(11;9)(q23;p22p23). Leukemia 11, 1469-1477, doi:10.1038/sj.leu.2400768 (1997).

Fine Needle Aspirate Serial Tumor Sampling Services To Boost Your Xenograft Study Capabilities

Hera Admin on December 1, 2020

Xenograft tumors from human cancer cell lines in immunocompromised mice are a staple of pre-clinical oncology research. However, tumor size limits, small blood volume, and minimal toxicology capabilities are significant challenges when using mice as an animal model. Hera BioLabs is proud to offer improved preclinical study opportunities using the SRG rat (OncoRat®), with tumor volumes growing up to 10,000+ mm³ or ten times the size of a mouse xenograft tumor. With larger tumor sizes, serial biopsy by fine needle aspirate (FNA) analysis can be completed to study tumor biology over time, all in the same animal and without inhibiting tumor growth.

The OncoRat® – A Perfect Complement For FNA Analysis

Similar to the NSG mouse, the OncoRat® lacks B, T, and NK cells, enabling efficient human tumor establishment. Extensive xenograft studies in the OncoRat® have shown improved engraftment efficiencies, tumor growth, size, and morphology compared to similar mouse models. Using the OncoRat®, toxicology, safety and pharmacokinetic/pharmacodynamic studies can be done in the same animal, while allowing for increased blood draws and larger tumor volumes that aid analysis after excision.

FNA Analysis Can Make Your Oncologic Study Data More Robust

In addition to post-tumor excision benefits, the large tumor size supported by the OncoRat® is ideal for serial tumor sampling while the animal is still undergoing treatment. Adding FNA data to your research will increase analysis opportunities for protein biomarkers, histology analysis, immune cell infiltration, RNA biomarkers, and RNA-seq to discover novel gene expression changes as the tumor is treated. These FNA analysis tools enable researchers to study tumor biology without sacrificing the animal, therefore decreasing costs and the number of animals associated with your study.

Moreover, serial FNA sampling will deliver the most complete picture of changes occurring inside the tumor as they are treated, not from a sole timepoint at the end of the study. Hera Biolabs has shown that up to two FNA samples can be taken weekly without affecting tumor growth (Figure 1), so one sample can go to histology and the other towards RNA/protein analysis!

Flexible Options for Your Unique Study

Hera BioLabs has grown successful xenograft tumors in the SRG OncoRat® from over 25 cell lines in a variety of cancers. FNA analysis has been done in-house with no negative effects on VCaP prostate cancer cells (Figure 1A), Daudi Burkitt’s Lymphoma cells (Figure 2), and H358 Non-Small Cell Lung Cancer (NSCLC) cells (Figure 3A). With extensive cell models available, Hera can sample the tumor with FNA throughout treatment using your drug or chemotherapy of choice, enabling thorough pre-clinical data in a minimal number of animals. FNA sampling is available at any time point of your choice after dosing – we can help you measure RNA changes 12 or 24 hours after treatment, protein changes via western blot, histological tumor microenvironment changes, or any time-sensitive parameter that your unique study requires. Successful western blots have been completed by clients on FNA samples collected at Hera, with positive results demonstrated for different proteins of interest (Figure 1B).

Samples obtained by FNA retain an extremely high cellular viability even after overnight shipping to your research lab. Independent analysis by a third party CRO has found that up to 96% of cells from the aspirate are viable after shipping (Figure 3B). Retaining cellular viability allows seamless off-site analysis at either your institution or an independent organization, including assays done on living cells. Of course, aspirates can also be flash frozen or fixed before shipment to best prepare the sample for your individual downstream research needs.

Contact Hera today to improve your preclinical xenograft studies with increased data collection through FNA sampling. Our OncoRat® model is specifically designed for multiple preclinical uses, marked by toxicology capabilities, pharmacokinetic studies, and increased tumor volumes that easily support FNA analysis to aid your research study needs.

Figure 1: (A) FNA sampling does not significantly affect tumor growth kinetics of VCaP prostate cancer cells injected subcutaneously into the flank of the SRG OncoRat®. Control (no FNA), one core (one FNA), and two cores (two FNA from the same tumor) samples were taken weekly at points indicated with black arrows. Each group is representative of three animals, and error bars indicate SEM. (B) Western blot analysis of FNA were assessed for prostate specific antigen (PSA), androgen receptor (AR), and vinculin expression.

Figure 2: FNA sampling does not have a significant effect on tumor growth kinetics of Daudi Burkitt’s Lymphoma cells injected subcutaneously into the flank of the SRG OncoRat®. FNA group (n=3) underwent weekly biopsies at points indicated with black arrows, non-FNA group (n=5) did not undergo sham procedure. Error bars indicate SEM. No statistical significance between groups as determined by unpaired t-test.

Figure 3: (A) H358 Non-Small Cell Lung Cancer (NSCLC) cells injected subcutaneously into the flank of the SRG OncoRat® have persisting large tumor volumes while undergoing FNA procedures. Tumors were sampled via FNA at points indicated with black arrows. Groups are representative of three animals, and error bars indicate SEM. (B) FNA samples retain very high cellular viability. Samples from (A) were obtained, placed in 1mL RPMI-1640 + 10% FBS and shipped overnight at 4°C for third party analysis. Each collection consists of one FNA sample from three xenograft tumors, error bars indicate SEM.

Contact Us to Learn More

If you would like to explore how the SRG Rat can help accelerate your oncology research, please contact us here.

 

References

  1. Noto, Fallon K., et al. “Abstract B067: A Rag2/Il2rg double-knockout rat (SRG OncoRat) enables precision-medicine based cancer studies with cell line-and patient-derived xenografts.” (2019): B067-B067.

Gene Editing With piggyBac: Creating A Curative Treatment For Beta-Thalassemia

Jack Crawford on October 21, 2020

Genetic Engineering Can Soon Lead To Lifelong Cures For Diseases That Were Previously Incurable – And ß-thalassemia Is The Perfect Example.

Through novel therapeutic methods like cell therapy, scientists have found a way to cure a disease that used to only be manageable. Previous gene editing methods used to treat ß-thalassemia were limited and nonspecific; however, advances like piggyBac and CRISPR-Cas9 based gene editing tools are opening doors for better patient outcomes.

What Is ß-Thalassemia?

Beta-thalassemia is a heritable genetic disease caused by a mutation in the hemoglobin beta (HBB) gene. Inheriting a mutated HBB gene leads to a lower production of hemoglobin.
Decreased hemoglobin is a problem because red blood cells, which carry hemoglobin, are used to transport oxygen throughout the body, so patients diagnosed with ß-thalassemia are often anemic. Patients with severe symptoms typically require invasive and frequent treatments with frequent blood transfusions and iron chelation therapy.

Previously, one patient diagnosed with ß-thalassemia had been treated successfully by having lentiviral delivery of a normal HBB gene into his hematopoietic stem and progenitor cells (HSPC). While successful for this patient, there is a high risk of off-target integration that makes lentiviral delivery an undesirable treatment option.

Viral vector directed gene therapies can be harmful, usually through insertion into multiple sites of the host genome. Problems in immune response could occur, and there remains a possibility of insertional mutations.

Steps To A Cure Through Next Generation Gene Editing

An improved ß-thalassemia treatment would correct the underlying mutation of the HBB gene. Correction of the HBB gene would allow for normal production of hemoglobin and ultimately better oxygen transport.

Using fibroblasts collected from the same patient, it is possible to generate induced pluripotent stem cells (iPSCs), which are then modified to carry the corrected form of the HBB gene. These cells could then be introduced back into the patient as HSPCs, curing the patient.

The bacteria derived RNA-directed Cas9 nuclease, also known as CRISPR-Cas9, can perform double stranded breaks in DNA in a directed manner. This makes room in the genome for insertion of desired genes. After the break is performed, the CRISPR-Cas9 technology can be combined with piggyBac in iPSCs to correct the HBB gene mutation.

How Does piggyBac Work?

The piggyBac system is a gene editing technology that can insert and remove genetic cargo from cells. This is done through transposase activity, allowing for the target gene to be cloned into a transposon vector and inserted into the region of interest.

PiggyBac has been shown to have little to no unintentional downstream effects, and is able to insert and remove genetic material without leaving behind any kind of “footprint” – meaning only the desired gene is inserted. Alternatively, the Cre/loxP system, another tool to insert/remove genes, leaves behind 34 bp that are foreign to the cell.

In the case of ß-thalassemia, piggyBac is able to remove the selection cassette and all foreign sequences, leaving behind only the corrected HBB gene and restoring normal hemoglobin activity.

Gene Editing Tools That Can Be Used With piggyBac

To use piggyBac to its full potential, it can be used in combination with gene editing tools that cause double stranded breaks in DNA. These tools are all nuclease based, meaning the breaks are caused by an enzyme.
There are three comparable DNA cleaving methods that target specific sites within DNA, which are:

  • Zinc finger nucleases (ZFN)
  • Transcription activator-like effector nucleases (TALENs),
  • Cas9 nucleases

Of these, Cas9 is the most reliable, readily available, and easiest to synthesize.

ZFNs have been known to be both expensive and difficult when it comes to their design, making them difficult to acquire for research.

TALENs are inefficient and difficult to validate, because many pairs are needed to ensure their cutting efficiencies.

Cas9 nucleases are efficient when used in systems such as CRISPR-Cas9, but off-target effects can be a limiting factor. However, Hera has built on the CRISPR-Cas9 platform to create the precise genome editing tool Cas-CLOVER which functions with no observable off-target effects.

Learn More About piggyBac, Cas-CLOVER And Hera BioLabs

Here at Hera, we use our proprietary gene editing system, Cas-CLOVER, which expands on the CRISPR-Cas9 platform for more accurate gene editing. Unlike CRISPR-Cas9, which is directed by one guide RNA, Cas-CLOVER has a dimeric guide system with one guide RNA and one Cas9 protein on each side of the target site. Because of its dimeric nature, Cas-CLOVER is more accurate than CRISPR-Cas9 and results in no observable off-target effects.
Moreover, Cas-CLOVER falls under a different set of patents than CRISPR-Cas9 which allows for hassle-free licensing agreements. Hera provides access to the Cas-CLOVER technology for drug discovery and early development.

Hera also provides licenses to piggyBac, which can be combined with Cas-CLOVER for scarless gene editing.
Whether you’re performing basic research or developing curative treatments like those for ß-thalassemia, Hera is ready to work with you to push your science forward.

To learn more about Hera how Hera utilizes the piggyBac transposase system click here. If you’d like to discuss licensing and forming a partnership reach out to the Hera team today by clicking here.

References

Xie, F., Ye, L., Chang, J. C., Beyer, A. I., Wang, J., Muench, M. O., & Kan, Y. W. (2014). Seamless gene correction of β-thalassemia mutations in patient-specific iPSCs using CRISPR/Cas9 and piggyBac. Genome research, 24(9), 1526–1533. https://doi.org/10.1101/gr.173427.114

The OncoRat® Is The Ideal Host For Patient-Derived Xenografts Of Ovarian Cancer Cells

Jack Crawford on December 11, 2018

Ovarian cancer is the most lethal gynecological cancer in the United States. Advances in cytotoxic, platinum-based chemotherapeutics combined with tumor resection surgery allows approximately 80% of these patients to achieve remission. Unfortunately, the vast majority have a tumor recurrence within 12-24 months and relapsed ovarian cancer is recognized as being universally incurable1-2.

Large genomic analyses of ovarian tumors, using databases including The Cancer Genome Atlas (TCGA), have revealed that ovarian tumors are highly heterogeneous. Specifically, no over-represented, targetable oncogenic mutations were revealed. Thus, alternative strategies must be employed to identify targetable driver pathways and sources of drug resistance in ovarian tumors1.

Ovarian tumors have a high degree of cell-population heterogeneity and also contain populations of cancer stem cells (CSCs) that contribute to growth and drug resistance in these cancers. It has been demonstrated that exposure of ovarian cancer cells to chemotherapeutics induces a gene expression program increasing cell-stemness, including the expression of CSC marker. For this reason, it is incredibly important that ovarian tumor models mimic the disease physiology in the patient as much as possible.

To leverage and study the natural heterogeneity of ovarian tumors, the DiFeo lab, lead by Dr. Analisa DiFeo, took resected high-grade serous ovarian cancer (HGSOC) tissue from a patient and established a patient-derived xenograft in a murine host – designated OV81. The importance of OV81 is that HGSOC tumors make up around 70% of the ovarian tumors diagnosed. Additionally, OV81 is cisplatin-naïve, so the tumor landscape is unchanged by chemotherapeutic treatment and the tissue taken is the best representation of the patient’s tumor.

From this patient-derived xenograft, the DiFeo lab isolated a cell line for in vitro study, designated OV81.2. OV81.2 cells have been used to identify some of the mechanisms of chemo-induced stemness, the mechanisms of drug resistance development, and metabolic changes that are unique to chemo-resistant ovarian cancer cells1-3. Having OV81.2 cells derived from a chemo-naïve ovarian tumor is paramount to identifying the mechanisms that define drug resistance.

Further examination of drug resistance development will require study replication and expansion into an in vivo xenograft model. OV81.2 cells were implanted into the OncoRat® and NSG mice. After three weeks of growth, the tumor xenografts in the OncoRat had grown to volumes nearly ten-fold higher than the NSG mouse. This demonstrates that the OncoRat is the ideal xenograft host for OV81.2 cells for further preclinical study of this important cell line.

Hera - Blog - The OncoRat® is the ideal host for patient-derived xenografts of ovarian cancer cells - Figure 1

References

  1. Wiechert, A.; Saygin, C.; Thiagarajan, P. S.; Rao, V. S.; Hale, J. S.; Gupta, N.; Hitomi, M.; Nagaraj, A. B.; DiFeo, A.; Lathia, J. D.; Reizes, O., Cisplatin induces stemness in ovarian cancer. Oncotarget 2016, 7 (21), 30511-30522.
  2. Hudson, C. D.; Savadelis, A.; Nagaraj, A. B.; Joseph, P.; Avril, S.; DiFeo, A.; Avril, N., Altered glutamine metabolism in platinum resistant ovarian cancer. Oncotarget 2016, 7 (27), 41637-41649.
  3. Nagaraj, A. B.; Joseph, P.; Kovalenko, O.; Singh, S.; Armstrong, A.; Redline, R.; Resnick, K.; Zanotti, K.; Waggoner, S.; DiFeo, A., Critical role of Wnt/β-catenin signaling in driving epithelial ovarian cancer platinum resistance. Oncotarget 2015, 6 (27), 23720-23734.

Understanding Transporter-Mediated Drug-Drug Interactions Using Humanized Liver Mice

Jack Crawford on January 31, 2018

Pharmaceutical researchers and regulators are increasingly aware of the influence that transporters have on the pharmacokinetics of drugs. These transporter mediated drug-drug interactions (DDIs) can precipitate adverse reactions in patients and as a result, safety guidelines across the United States, Europe and Japan have been updated to require safety assessments of DDIs in large scale clinical trials of pharmaceuticals.

Patient drug interactions related to hepatic organic anion-transporting polypeptides (OATPs) are of particular concern when evaluating statins. According to a recent article by Uchida, et al published in Drug Metabolism and Disposition, the systemic exposure of rosuvastatin, a drug widely used to treat elevated cholesterol levels, was “significantly increased by concomitant dosing with cyclosporine A, and commonly lower dosage of rosuvastatin is therefore required when administered with cyclosporine A” in a clinical environment.

Previously, in vivo animal model studies have been conducted to predict DDIs in humans, but the reality is that often times little correlation exists between preclinical murine outcomes and actual clinical outcomes in patients.  To combat this, “knockout models lacking murine oatp isoforms and OATP-humanized transgenic mice have been developed” notes Uchida, et al; however, the pharmacokinetics may still be affected by other inherent murine transporters.

As such the researchers in this study examined the effectiveness of using PXB-mice (chimeric mice with humanized livers and high levels of human hepatocytes) to predict human DDIs between rosuvastatin and cyclosporine A.  The “observed DDIs in vivo were considered to be reasonable,” according to Uchida, et al, and suggest that humanized liver mice such as PXB and TK-NOG mice could play an important role in drug discovery and the evaluation of DDIs in a preclinical setting.

Humanized Mouse Model Allows For Study Of Immune Response To Decellularized ECM Biomaterials

Jack Crawford on June 23, 2017

Humanized rodent models have been used extensively for studying autoimmune diseases, viral infections, xenogeneic transplantation and, more recently, allogenic stem cell transplantation; however, until now, researchers have not used these murine models for studies in the field of biomaterials. In their recent paper published in Biomaterials, Wang et al used a humanized mouse model to “assess the human immune response to decellularized extracellular matrix (ECM) biomaterials, specifically injectable hydrogels derived from porcine or human myocardium, which Biomaterials, Wang et al used a humanized mouse model to “assess the human immune response to decellularized extracellular matrix (ECM) biomaterials, specifically injectable hydrogels derived from porcine or human myocardium, which were initially developed to treat the heart post-myocardial infarction.”

This is important because the rodents typically used for biocompatibility testing provide limited representation of the human immune response because of differences in immune cell receptors, cytokine expression and response to various stimuli highlight how responses in rodents might not correlate with outcomes observed in humans. “Immune cells in the humanized mouse model, particularly T-helper cells, responded distinctly between the xenogeneic and allogeneic biomaterials,” according to Wang et al. “The allogeneic extracellular matrix derived hydrogels elicited significantly reduced total, human specific, and CD4 T-helper cell infiltration in humanized mice compared to xenogeneic extracellular matrix hydrogels, which was not recapitulated in wild type mice.”

Although this model certainly still has some limitations, the study was successful and gives high hopes to improving outcomes in the field of biomaterials, which continue to play an integral role in applications related to wound healing, hernia repair, skeletal muscle defect repair and hear attacks. As suggested by Wang et al, “decellularized ECM biomaterials are an attractive platform for biomaterial therapies since tissue derived from ECM can promote tissue remodeling by influencing cellular metabolism, proliferation, migration, maturation and differentiation” and these humanized mouse models will allow for further study of human immune cell responses to biomaterials in an in vivo environment.

Chimeric Humanized Mouse Models: Understanding Human And Mouse Cell Interactions

Jack Crawford on February 28, 2017

Humanized liver mouse models are increasingly being used in preclinical trials and have allowed for groundbreaking in-vivo research to evaluate everything from human-specific drug toxicity and efficacy to gene therapies. Unlike their transgenic mouse model counterparts, chimeric liver mouse models that include human hepatocytes and it is important for researchers to better understand the interactions between the implanted human cells and native mouse cells especially for drug metabolism studies.

In a recent study by Chow et al published in The Journal of Pharmacology and Experimental Therapeutics, it was shown that as a result of the species mismatch between human and mouse cells certain deficiencies are increasingly common, including dysregulation of hepatocyte proliferation and bile acid homeostasis in hFRGN livers that led to hepatotoxicity, gallbladder distension, liver deformity and other extrahepatic changes. “Although the nuclear receptors in human and other species share common targets, species difference in nuclear receptor activation exists”, and Chow et al suggest that additional research may be necessary to fully understand the inter-organ communication between human and mouse organs in h-chimeric mice.

Specifically, the miscommunication between human hepatocytes and murine stellate cells (which typically signal to hepatocytes to stop proliferating) is an important consideration. When this occurs, intracellular spaces are frequently reduced and cholangiocyte growth is inhibited, which can result in reduced bile flow as well as increased bile acid accumulation and toxicity.
Although we do not believe that any of these factors are reason enough to discontinue the use of humanized mouse or rat models for preclinical research, Chow et al do point out the need for increased awareness and the importance of addressing these deficiencies when reporting data in human drug metabolism studies.

Transposon Mutagenesis Helps Identify Genes Mediating Drug Resistance In The Treatment Of CLL

Jack Crawford on February 28, 2017

First time treatment of Chronic Lymphocratic Leukemia (CLL) generally requires a chemotherapy regimen that includes fludarabine. And although this potent drug combination has an astounding overall response rate of more than 90%, the unfortunate reality is that most patience will eventually relapse. And even more concerning is that in addition to the relatively small percentage of patients who are inherently resistant to fludarabine treatments from the start, with each subsequent use of this chemotherapy cocktail, data shows increased patient populations with acquired resistance to fludarabine-based chemotherapy, which ultimately presents a significant challenge for long-term disease control.

In order to better understand which specific genes and genetic pathways are mediating fludarabine resistance, Pandzic et al performed a “piggyBac transposon mutagenesis screen in a human CLL cell line” and their findings were recently reported in Clinical Cancer Research. In this study, a CLL cell line was subjected to random mutagenesis through integration of piggyBac transposons into genomic DNA. The cells were screened for resistance to fludarabine and the insertion sites of the piggyBac transposons identified through the use of Splinkerette PCR (spPCR). This screen not only revealed known resistance mediator genes, such as DCK (deoxycytidine kinase), but it also identified three novel genes, including BMP2K, which was shown to modulate response to fludarabine, although it had previously not been linked to CLL patients with fludarabine resistance.

This study demonstrated that piggyBac transposon mutagenesis screens have the ability to help lead the way when it comes to identifying genes that mediate patient sensitivity to specific drug therapies, including, but not limited to fludarabine-based chemotherapy. The piggyBac transposon is one of Hera BioLabs core technologies and we can assist with establishing various genome engineering and mutagenesis screens utilizing piggyBac in combination with CRISPR/Cas9.