Hera Admin – Hera BioLabs

How the piggyBac^® Transposon Sheds Light on Breast Cancer Drug Resistance

Hera Admin on May 30, 2023

With over 2.2 million annual cases, breast cancer is the most common cancer in women, but drug resistance remains a formidable challenge, particularly due to the development of drug resistance in recurrent or metastatic cases.¹ The activation of the phosphatidylinositol 3-kinase (PI3K) pathway, driven by mutations in the PIK3CA gene, is a common oncogenic driver and plays a pivotal role in breast cancer development.²

Alpelisib, a PI3Kα-selective inhibitor, has shown promise in treating patients with PIK3CA-mutated breast cancer. However, studies have shown that certain mutations confer resistance to alpelisib, including upregulated retinoblastoma protein (Rb) and loss of phosphatase and tensin homologue protein (PTEN).³

Crucial Role of the piggyBac Transposon

In this context, a new study has harnessed the power of the piggyBac transposon system to unravel these key resistance mechanisms and propose a novel therapeutic strategy.

Auf der Maur and colleagues performed a genome-wide transposon-mediated mutagenesis screen using a mouse model of PIK3CAH1047R-driven mammary tumors.⁴ To do this, the team combined the piggyBac (PB) transposon system5 with a murine whey-acidic protein (WAP)-driven and PIK3CA^H1047R mutant mammary tumor model.⁶

The piggyBac system allowed for the identification of NF1 loss, a negative regulator of RAS, as a resistance mechanism against PI3Kα inhibitors. The study further elucidated the metabolic changes associated with NF1 loss, revealing enhanced glycolysis and lower levels of reactive oxygen species (ROS) in cancer cells.

Figure 1. Illustration of the transgenic mouse lines used for the in vivo transposon mediated resistance screen.

By leveraging the piggyBac transposon system, this study has shed light on the mechanisms underlying resistance to PI3Kα inhibitors in breast cancer. The identification of NF1 loss as a resistance mechanism opens new avenues for more effective therapeutic strategies. As such, the group observed synergistic anti-tumor effects when NF1 knockout cells were treated with N-acetylcysteine (NAC) along with a PI3Kα inhibitor.

These findings also highlight the importance of utilizing innovative genetic tools like piggyBac to unravel resistance mechanisms and propel advancements in breast cancer treatment. This research offers hope for improved treatment strategies and underscores the power of the piggyBac system in advancing our understanding of drug resistance in breast cancer.

Learn More About piggyBac

The piggyBac system offers a versatile and efficient approach for genome editing, with applications across diverse research fields. Its remarkable cargo capacity of over 200kb sets it apart from other transposon and viral delivery vehicles, and makes it the ideal choice for creating stable cell lines.

To learn more about piggyBac and how Hera can help accelerate your gene editing research with our toolkit, click here.

References

Sung H., Ferlay J., Siegel R.L., Laversanne M., Soerjomataram I., Jemal A., Bray F. Global cancer statistics 2020: GLOBOCAN estimates of Incidence and mortality worldwide for 36 cancers in 185 countries. CA. Cancer J. Clin. 2021; 71: 209-249. https://doi.org/10.3322/caac.21660
Miller T.W., Rexer B.N., Garrett J.T., Arteaga C.L. Mutations in the phosphatidylinositol 3-kinase pathway: role in tumor progression and therapeutic implications in breast cancer. Breast Cancer Res. 2011; 13: 224 https://doi.org/10.1186/bcr3039
André F., Ciruelos E., Rubovszky G., Campone M., Loibl S., Rugo H.S., Iwata H., Conte P., Mayer I.A., Kaufman B. et al. Alpelisib for PIK3CA -mutated, hormone receptor–positive advanced breast cancer. N. Engl. J. Med. 2019; 380: 1929-1940 https://doi.org/10.1056/nejmoa1813904
Auf der Maur, P., et al. (Year). N-acetylcysteine overcomes NF1 loss-driven resistance to PI3Kα inhibition in breast cancer. Cell Reports Medicine, 4(4), 101002.
Rad R., Rad L., Wang W., Cadinanos J., Vassiliou G., Rice S., Campos L.S., Yusa K., Banerjee R., Li M.A. et al. PiggyBac transposon mutagenesis: a tool for cancer gene discovery in mice. Science. 2010; 330: 1104-110 https://doi.org/10.1126/science.1193004
Meyer D.S., Brinkhaus H., Müller U., Müller M., Cardiff R.D., Bentires-Alj M. Luminal expression of PIK3CA mutant H1047R in the mammary gland induces heterogeneous tumors. Cancer Res. 2011; 71: 4344-4351 https://doi.org/10.1158/0008-5472.CAN-10-3827

Advantages of Utilizing the SRG rat for Investigating Tumor Metastasis

Hera Admin on March 28, 2023

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The dissemination of tumor cells into systemic circulation is a critical area of cancer research, but it presents significant challenges. One obstacle is the lack of appropriate animal models that can support larger tumors and enable frequent, large volume blood collection. In this article, we explore the innovative research conducted by Ami Yamamoto and colleagues in Kevin Cheung’s lab at Fred Hutchinson Cancer Center.

Challenges with in vivo modeling of breast cancer dissemination

Aggressive cancers frequently exhibit necrotic tumor interiors, which is associated with poor clinical prognosis and the development of metastasis. ^[2-6] Despite this association, the precise mechanisms by which the necrotic core promotes metastasis are not fully understood. In this study, the research team employed SRG rats as a model organism to improve the detection of dissemination events and study molecular mechanisms that mediate the relationship between tumor necrosis and metastasis. ^[1]

Supplemental Figure S1: Orthotopic transplantation into rats produce 3x larger tumors, 10x more CTCs, and 4x more lung metastases than into mice.

Breakdown of Figure S1:

S1A) Experimental schema. 4T1 mouse mammary tumor cells labeled with membrane GFP (4T1-GFP) were orthotopically transplanted into a single #4 mammary fat pad of SRG rats (n=6) or NSG mice (n=24). Animals were sacrificed at 24 days post-transplantation.
S1B-C) Final tumor weight and estimated final tumor volume per animal.
S1D) Blood volume collected per animal.
S1E) Representative micrographs of single CTCs and CTCs clusters from 4T1-GFP transplanted SRG rats. GFP denoted in green. DAPI in blue.
S1F-G) Single CTC and CTC cluster abundance per animal. Individual blood samples from 8 mice were pooled into one tube for a total of n=3 samples. Events per individual animal are reported.
S1H) Percentage of CTC events that were single CTCs or CTC clusters. Mean ± SD.
S1I) Representative stereomicroscope images of lung metastases from NSG mice and SRG rats.
S1J) The number of lung metastases determined by stereomicroscopy in transplanted SRG rats and NSG mice.
Mean values shown on graphs. All P-values determined by Welch’s t-test.

Yamamoto et al. began by comparing tumor growth and metastasis in SRG rats versus NSG mice. They implanted the same volume of luciferase-tagged 4T1 mouse mammary carcinoma cells (4T1-Luc) into the mammary tissue of SRG rats or NSG mice. After 24 days of growth, as tumors approached the maximum allowable size for mice, tumors were removed and characterized. Rat tumor mass and volume was three-fold higher than mouse (Figure 1A-C). Additionally, the team was able to collect ten-fold more blood from rats versus mice, leading to a proportional ten-fold increase in the recovery of both single circulating tumor cells (CTCs) and CTC clusters (Figure 1D, F, & G). The ratio of CTC clusters to single CTCs did not differ between rats and mice, nor did their morphological appearance (Figure 1E & H). In keeping with higher numbers of CTCs, rats exhibited more lung metastases than mice, assessed using stereomicroscopy for GFP (Figure 1I & J). The authors concluded that, because the same number of 4T1 cells was transplanted in both SRG rats and NSG mice and because tumors were harvested at the same time, our direct comparison showed that the rat transplantation model increases the efficiency of detecting rare tumor dissemination events by 10-fold.

Having established the utility of the SRG rat, the authors proceeded to show that the development of necrosis is temporally related to dissemination of CTCs, in both rats and humans. Importantly, they report that dissemination occurs in the dilated blood vessels surrounding the necrotic tumor core, and that dissemination events are dependent on ANGPTL7 that is produced by tumor cells bordering the necrotic region. The authors suggest that ANGPTL7 could be a viable pharmacological target for the inhibition of metastatic events.

Benefits of the SRG rat model in CTC research

These findings make the SRG rat a promising model for studying CTC dissemination, and these results provide valuable insights into the molecular mechanisms underlying tumor dissemination and pave the way for the development of new therapies for breast cancer.

Figure 4: The SRG Rat lacks B, T, and NK cells.

Learn more about Hera’s SRG Rat Model

The SRG rat model has emerged as a promising tool for studying circulating tumor cells and metastasis, providing several advantages over traditional mouse models. Hera offers in vivo oncology studies utilizing the SRG rat in a wide variety of xenograft and PDX tumor models, allowing researchers to gain valuable insights into the molecular mechanisms underlying tumor dissemination.

Contact Charles River Labs for more information.

References:

Yamamoto A, Huang Y, Krajina BA, et al. Metastasis from the tumor interior and necrotic core formation are regulated by breast cancer-derived angiopoietin-like 7. Proc Natl Acad Sci U S A. 2023;120(10):e2214888120. doi:10.1073/pnas.2214888120
Fisher, E. R., Sass, R., & Fisher, B. (1984). Pathologic findings from the National Surgical Adjuvant Project for Breast Cancers (protocol no. 4). X. Discriminants for tenth year treatment failure. Cancer, 53(3 Suppl), 712–723. https://doi.org/10.1002/1097-0142(19840201)53:3+<712::aid-cncr2820531320>3.0.co;2-i
Jimenez, R. E., Wallis, T., & Visscher, D. W. (2001). Centrally necrotizing carcinomas of the breast: a distinct histologic subtype with aggressive clinical behavior. The American journal of surgical pathology, 25(3), 331–337. https://doi.org/10.1097/00000478-200103000-00007
Leek, R. D., Landers, R. J., Harris, A. L., & Lewis, C. E. (1999). Necrosis correlates with high vascular density and focal macrophage infiltration in invasive carcinoma of the breast. British journal of cancer, 79(5-6), 991–995. https://doi.org/10.1038/sj.bjc.6690158
Bredholt, G., Mannelqvist, M., Stefansson, I. M., Birkeland, E., Bø, T. H., Øyan, A. M., Trovik, J., Kalland, K. H., Jonassen, I., Salvesen, H. B., Wik, E., & Akslen, L. A. (2015). Tumor necrosis is an important hallmark of aggressive endometrial cancer and associates with hypoxia, angiogenesis and inflammation responses. Oncotarget, 6(37), 39676–39691. https://doi.org/10.18632/oncotarget.5344
Zhang, L., Zha, Z., Qu, W., Zhao, H., Yuan, J., Feng, Y., & Wu, B. (2018). Tumor necrosis as a prognostic variable for the clinical outcome in patients with renal cell carcinoma: a systematic review and meta-analysis. BMC cancer, 18(1), 870. https://doi.org/10.1186/s12885-018-4773-z

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Addressing the limitations of chemotherapeutic drug delivery with engineered protein polymers

Hera Admin on January 24, 2023

The standard care of treatment for many types of cancer and infectious diseases around the world is currently chemotherapy. However, chemotherapies are widely known to be linked to severe toxicity and resistance that develop mostly due to non-specificity, leading to overall disease progression.

Figure 1b. Chemotherapeutics have poor specificity which results in higher doses of medication due to the lack of bioavailability. Higher doses result in increased toxicity to healthy cells, drug resistance, and various other side effects pictured here.

To combat these limitations, recent advancements in recombinant technologies have created some novel genetically engineered protein polymers such as Elastin-like polypeptide (ELP), Silk-like polypeptide (SLP), and hybrid protein polymers with specific sequences to impart consistent and precise features to target proteins, and which have given excellent preclinical results in studies so far.

The synthesis and administration of chemotherapeutics have thus made use of these protein polymers, overcoming many of the limitations of traditional therapeutics.

The research team responsible for a recent publication, summarizes the development of such advanced recombinant protein polymers, and discusses the main challenges associated with designing targeted delivery systems for chemotherapeutics, as well as the potential for unique gene editing platforms like Hera’s Cas-CLOVER to further enhance their applications in the future.

Challenges in traditional chemotherapeutics processing with recombinant protein polymers

Recombinant protein polymer research has grown significantly in popularity over the past few decades mainly because it has addressed a number of problems with traditional drug delivery systems. Many of these new polymers were discovered to be highly effective at delivering therapeutic payload, and some of them have even received FDA approval.

Natural protein polymers are utilized in forms such as nanoparticles, micelles, scaffolds, and hydrogels as the vehicles for targeted drug delivery to tissues.

See Figure 3:

a) Micelles
b) Hydrogel
c) Micelle containing targeting moiety and chemotherapeutics
d) Bi-functional micelle chemotherapeutics formulation
f) Modified liposomes with chemotherapeutics
g) Hybrid hydrogel synthesized by recombinant protein polymers and other protein polymers, like collagen, encapsulating chemotherapeutics.

Figure 3. Recombinant protein polymer-based drug delivery platforms are formulated into various types of nanoplatforms and are formulated to chemotherapeutics.

Nevertheless, despite their effectiveness, there are a number of significant issues with these current systems that must be resolved.
Natural protein polymers are currently widely available, manageable, and affordable for use in a variety of biological applications, but they still have some drawbacks. For example, batch-to-batch deviations in raw materials can render the final products being prone to efficacy variance. Additionally, the molecular weight of the protein polymers limits their usage to only a few dozen applications at this time.

Figure 1a. The molecular weight distribution and solubility of chemotherapeutics shows that most drugs have lower molecular weight and increased hydrophobicity.

The future of recombinant protein polymers and targeted drug delivery systems

In response to these and other problems, genetic engineering has been used to develop recombinant protein polymers. These new polymers are synthesized using identical sequences and scaled up for large-scale production, thus resulting in protein polymers without as much batch-to-batch variance as before.

Figure 2. A representation of recombinant protein polymer synthesis and its formulation with chemotherapeutics. The recombinant protein is isolated, purified, and combined with chemotherapeutics to form recombinant protein-chemotherapeutics formulation.

This technology offers new strategies for controlling molecular weight and the overall structure of the target protein. Additionally, other targeting sequences can be further fused with the protein for advanced drug delivery applications in the near future. The team in this study mentioned Cas-CLOVER as a great example for this application, as it has the potential to revolutionize the world of therapeutic bioprocessing by making precise, intentional and beneficial edits in the platform bioprocessing cell lines resulting in efficient and effective developmental control.

Accelerate your protein polymer formulations and bioprocessing projects with Hera’s Cas-CLOVER technology

Cas-CLOVER is a highly specific system demonstrating 99%+ efficiency, and it’s covered by issued patents with clear commercial access. To learn more about Cas-CLOVER, and how Hera can help accelerate your cell line development studies, click here. We have everything for your research needs, including flexible licensing options.

References:

Anjali Phour, Vidit Gaur, Ahana Banerjee, Jayanta Bhattacharyya. Recombinant protein polymers as carriers of chemotherapeutic agents. Advanced Drug Delivery Reviews, Volume 190 (2022), https://doi.org/10.1016/j.addr.2022.114544.

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Cas-CLOVER achieves targeted knock-ins in induced pluripotent stem cell lines

Hera Admin on November 17, 2022

As cell line development bioprocessing turns from traditional random plasmid integration to targeted knock-ins for maximizing yield and stability, it is important to choose the right gene editing tools. The RNA-guided Cas9 nuclease from Streptococcus pyogenes (SpCas9), which is the most commonly used enzyme in genome-editing applications, can yield significant levels of off-target activity, with reported off-target cutting as high as 13%. Multiplex editing with SpCas9 for cell line engineering can also lead to high rates of translocations, up to 4% for individual translocations or rearrangements and up to 5% cumulatively.¹

A recent study published in Molecular Therapy Nucleic Acids saw the authors at Poseida Therapeutics deploy the dual RNA-guided dimeric endonuclease Cas-CLOVER, as an alternative to CRISPR/Cas9. Hera and Poseida both use Cas-CLOVER technology in different fields of use. Let’s take a closer look at the targeted knock-in data in this study with Cas-CLOVER.

Comparison of knockout and knock-in success in iPSCs between Cas-CLOVER and Cas9

In this study, the team determined HDR (homology-directed repair)-mediated knock-in potential using Cas-CLOVER by examining reporter cassette delivery at several loci (GAPDH, B2M, and HBB) in induced Pluripotent Stem Cells (iPSCs).

Figure 1: Schematic showing targeting strategy.

¹ Cas-CLOVER is a novel high-fidelity nuclease for safe and robust generation of TSCM-enriched allogeneic CAR-T cells. Madison et al.Molecular Therapy – Nucleic Acids. August 8, 2022.

At most loci, Cas-CLOVER’s indel – as well as knock-in – efficiency is equal to or greater than that of Cas9, as demonstrated in the below example for B2M.

Figure 2: Quantification of indel (left) and HDR rates (right) at the B2M locus.

At HBB, knockin efficiency was higher using Cas-CLOVER, but the difference was probably not due to differential efficiency of target cleavage, as gauged by Cas-CLOVER’s lower indel generation (see image below). Rather, the higher efficiency was likely a result of the larger deletions, with overhangs, that are characteristic of Cas-CLOVER. Cas9 indels generally result in small 1-3 bp blunt-ended cuts.

Figure 2: Quantification of indel (left) and HDR rates (right) at the HBB locus. Flow for HDR measurement at the HBB locus was performed 14 days post-nucleofection to ensure episomal GFP expression from the donor plasmid had decreased below the detection limit.

After Cas-CLOVER editing, potential off-target sites were queried for the presence of indel mutations. Indel frequency at off-target sites did not occur at a statistically significant level above background. The team concluded that, within primary T cells from multiple donors, the Cas-CLOVER platform performs as well as any other targeted nuclease, but with much lower off-target activity. Notably, Cas-CLOVER yields efficient multiplexed gene editing.

Targeted knock-in capability is becoming essential for cell line development

Induced pluripotent stem cells (iPSCs) are being used more and more in therapeutic bioprocessing and in synthetic biology. Moreover, the level of targeting that the Poseida team was able to achieve with Cas-CLOVER is relevant for any cell line development professional – including those engineering custom cells for preclinical research purposes.

Learn more about Hera’s Cas-CLOVER and piggyBac technology

To learn more about Cas-CLOVER, piggyBac, and how Hera can help accelerate your gene editing research with our toolkit, click here. We have everything for your gene editing needs, including flexible front or back-loaded service terms and licensing options. Access our technology as quickly as possible by shopping online today.

References

Cas-CLOVER is a novel high-fidelity nuclease for safe and robust generation of TSCM-enriched allogeneic CAR-T cells. Madison et al.Molecular Therapy – Nucleic Acids. August 8, 2022.

piggyBac Case Study: Generating Full-length NKR-P1 Transfectants

Hera Admin on October 19, 2022

The high efficiency and stability of piggyBac integration have been proven in a variety of recent studies. With a large cargo capacity of over 200kb, piggyBac surpasses many other delivery technologies, making it the optimal choice for stable cell line creation. Hundreds of peer-reviewed papers have been published as a result of the piggyBac platform.

In one published study, full-length NKR-P1 transfectants were generated using the piggyBac system. Let’s take a closer look at the critical role that piggyBac played in helping the research team build a comprehensive model of NKR-P1-LLT1 interactions to better understand how they function in immune-related diseases and cancer.

Human NKR-P1:LLT1 complex reveals clustering in the immune synapse

Innate immune lymphocytes known as natural killer (NK) cells have a variety of activating and inhibiting surface receptors, allowing them to recognize and kill malignant and infected cells. One of these is NKR-P1, which acts as an inhibitory receptor. Additionally, NK cells support the body’s adaptive immune response and even preserve a type of immunological memory, further highlighting the vital functions of these cells in immunity.

LLT1, a ligand of NKR-P1, is usually expressed on monocytes and B cells and helps maintain NK cell self-tolerance. However, it is also upregulated in many cancers, allowing those cancer cells to evade the immune system by binding to NKR-P1, which inhibits the NK cells.

Figure 1: Depiction of the hypothetical arrangement of NKR-P1 dimers (cyan and blue) interacting with LLT1 dimers (green and yellow). Bláha et al., 2022.

LLT1 ligation induces NKR-P1 receptor clustering

In this study, full-length NKR-P1 transfectants were generated using the piggyBac system, and induced to express the receptor in a limited density to allow single-molecule localization microscopy. Unlike other gene editing platforms, piggyBac allows you to integrate large or small transgenes seamlessly. Demonstrated to work efficiently beyond 250kb+ in size, Hera regularly creates vectors between 10kb and 25 kb including multi-component ion channels and GPCRs in a single construct.

Super-resolution dSTORM microscopy shines with piggyBac

For the single-molecule localization microscopy, full-length NKR-P1 stable transfectants were generated in HEK293S GnTI– cell line using the piggyBac transposon-based system with doxycycline-inducible protein expression. It’s important to note that long-term stability is essential for producing consistent and reproducible results like this, and the piggyBac transposon genomic integrations are extremely stable for months of continuous culture and >50 passages.

Figure 2: piggyBac’s high carrying capacity allowed for the creation of full-length NKR-P1 stable transfectants in the HEK293S GnTI– cell line.

Successful Results using piggyBac Technology

The research team’s findings clarify the mode of signal transduction of the human NKR-P1 receptor within the NK cell immune synapse and how this receptor overcomes its low affinity for LLT1 by ligand binding-induced cross-linking and clustering, serving as a useful model for the future description of related homologous low-affinity complexes. By observing the clusters using SEC-SAXS analysis, dSTORM super-resolution microscopy, and freshly isolated NK cells, with the help of piggyBac technology, the team concluded that only the ligation of both LLT1 binding interfaces leads to effective NKR-P1 inhibitory signaling.

piggyBac Patented Technology for your Industry

The piggyBac Transposase/Transposon DNA Delivery System is a non-viral gene editing platform designed for stable integration and expression across a variety of industries and research. Hera BioLabs is the exclusive licensee and sublicensor of this patented technology for drug discovery and early development research including creating custom cell lines for R&D, therapeutic bioprocessing, animal model embryo engineering, and more.

Reach out to us to learn more about the piggyBac technology, the services we offer, and available licenses for your industry.

References

Bláha, J., Skálová, T., Kalousková, B. et al. Structure of the human NK cell NKR-P1:LLT1 receptor:ligand complex reveals clustering in the immune synapse. Nat Commun 13, 5022 (2022). https://doi.org/10.1038/s41467-022-32577-6

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

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

Cancer Facts & Figures 2022, https://www.cancer.org/cancer/acute-myeloid-leukemia/about/key-statistics.html#references (2022).
Almosailleakh, M. & Schwaller, J. Murine Models of Acute Myeloid Leukaemia. Int J Mol Sci 20, doi:10.3390/ijms20020453 (2019).
Project Optimus: Reforming the dose optimization and dose selection paradigm in oncology, https://www.fda.gov/about-fda/oncology-center-excellence/project-optimus (2022).
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).
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).

Rapid Creation of Bioluminescent Cell Lines for Orthotopic and Metastatic Rodent Oncology Studies

Hera Admin on February 25, 2022

The piggyBac (PB) transposon system delivers highly stable transgenes without gene silencing for over 40 passages, making PB superior to viral methods for creating reporter cell lines. The PB transposon is a mobile genetic element that is integrated into the host genome via the PB transposase “cut and paste” mechanism. To achieve stable genomic integration, The PB transposon is co-transfected with a PB transposase protein or expression vector. The transposase inserts the gene cargo and ITRs into the host genome at TTAA sites (Figure 1).

Figure 1: The piggyBac transposon system

Advantages to piggyBac in Bioluminescent Cell Line Creation

Hera BioLabs has used PB to generate numerous tumorigenic cell lines with robust luciferase expression. With previously validated cell lines, it takes Hera as few as 4 weeks to generate stable luciferase(+) cells for downstream use. These cell lines serve as an invaluable resource for monitoring tumor xenograft growth in either subcutaneous or orthotopic anatomical locations. In the subcutaneous space, in vivo luminescent imaging allows for assessment of primary tumor growth using the AMI HT from Spectral Imaging Instruments Imaging. For example, Hera recently made H358 non-small cell lung cancer cells with stable luciferase expression for subcutaneous inoculation in our SRG OncoRats and tracked tumor growth with luminescent imaging (Figure 2). Importantly, high luciferase expression confers adequate luminescent intensity for visualization of deep orthotopic tumors and metastases.

Figure 2: In vivo bioluminescent imaging using the AMI HT (Spectral Instruments Imaging). SRG OncoRats were given subcutaneous inoculations with 4 x 10⁶ NCI-H358-Luciferase cells and tumors grown for 5 weeks.

If your efficacy testing requires in vivo imaging or tracking metastases, contact us for studies in luciferase-expressing cell lines. Use one of our previously engineered lines or let us express luciferase in your cell line!

Learn more by contacting us today!

In Vivo Gene Delivery Using piggyBac With Hydrodynamic Tail Vein Injection

Hera Admin on August 19, 2021

The piggyBac (PB) transposon system, originally identified in the Cabbage Looper moth, is a mobile genetic element that can be integrated into a host genome via the PB transposase “cut and paste” mechanism. To perform stable genomic integration, piggyBac transposon must be co-transfected with a piggyBac transposase protein or expression vector. The transposase then inserts the gene cargo and ITRs into the host genome (also known as transposition) (figure 1)1 at TTAA sites. The piggyBac transposon system offers numerous advantages over other gene delivery systems, and it is frequently used to generate genetically modified stable cell lines and transgenic animal models.

Hydrodynamic tail vein injection (HTVI) is an efficient strategy for in vivo gene delivery to mouse liver. HTVI avoids some drawbacks associated with other gene delivery techniques: adeno-associated viral vector can be immunogenic and targeted liposome delivery often fails to deliver adequate amounts of gene cargo.

The technique involves rapid injection (<5 seconds) of a large volume of aqueous solution (8-10% volume/body weight) containing nucleic acids. In early studies, gene delivery using HTVI often failed to achieve clinically significant protein expression levels. However, several groups have worked on optimizing the technique to increase protein expression in mice.

Physiologically, hydrodynamic tail vein injection achieves delivery of large volumes of solution by causing back flow from the hepatic vein into the liver. Additionally, the quick increase in blood volume also causes transient cardiac arrythmia, which resolves within minutes due to the rapid heart rate in mice. Accordingly, HTVI does not work as well in larger animals that have slower heart rates, including rats2. At the cellular level, the exact mechanism of gene cargo delivery is unknown. However, increased endocytosis has been described when cells encounter hypo-osmotic stress and increases in endocytic vesicles have been reported in hepatocytes following HTVI3.

Several groups have used HTVI to deliver genes packaged in piggyBac to mouse liver. An early study reported robust luciferase expression for 6 months following HTVI delivery of piggyBac4. Another group, working with hemophilia A mice, reported that full length Factor VIII cDNA delivery resulted in stable expression of factor VIII protein for nearly 1 year, along with normalization of blood clotting5. Other studies have successfully shown luciferase expression for up to 300 days after injection (figure 2)6. This group also combined doxycycline-inducible gene expression with HTVI delivery of piggyBac and showed inducible gene expression for up to 120 days6.

Due to its ability to carry extremely large gene cargo (up to 200 kb pairs), the piggyBac platform is an extremely flexible technology capable of mediating long-term expression of simple to complicated gene editing systems. Paired with HTVI, it is an attractive option for manipulating gene expression in mouse liver.

References:

Sato, M., Inada, E., Saitoh, I., Watanabe, S. & Nakamura, S. piggyBac-Based Non-Viral In Vivo Gene Delivery Useful for Production of Genetically Modified Animals and Organs. Pharmaceutics 12, doi:10.3390/pharmaceutics12030277 (2020).
Sendra, L., Herrero, M. J. & Alino, S. F. Translational Advances of Hydrofection by Hydrodynamic Injection. Genes (Basel) 9, doi:10.3390/genes9030136 (2018).
Crespo, A. et al. Hydrodynamic liver gene transfer mechanism involves transient sinusoidal blood stasis and massive hepatocyte endocytic vesicles. Gene Ther 12, 927-935, doi:10.1038/sj.gt.3302469 (2005).
Doherty, J. E. et al. Hyperactive piggyBac gene transfer in human cells and in vivo. Hum Gene Ther 23, 311-320, doi:10.1089/hum.2011.138 (2012).
Matsui, H. et al. Delivery of full-length factor VIII using a piggyBac transposon vector to correct a mouse model of hemophilia A. PLoS One 9, e104957, doi:10.1371/journal.pone.0104957 (2014).
Saridey, S. K. et al. PiggyBac transposon-based inducible gene expression in vivo after somatic cell gene transfer. Mol Ther 17, 2115-2120, doi:10.1038/mt.2009.234 (2009).M

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

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.