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:
  1. 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  
  2. 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  
  3. 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 
  4. 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 
  5. 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
  6. 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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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).

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).

how-piggybac-works

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.

bioluminescent-rats

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!

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.