<img height="1" width="1" src="https://www.facebook.com/tr?id=1582471781774081&amp;ev=PageView &amp;noscript=1">
  • Menu
  • crown-logo-symbol-1-400x551

Find it Quickly

Get Started

Select the option that best describes what you are looking for

  • Services
  • Models
  • Scientific Information

Search Here For Services

Click Here to Start Over

Search Here For Models

Click Here to Start Over

Search Here For Scientific Information

Click Here to Start Over

In Vitro

Boost oncology drug discovery with XenoBase®, featuring the largest cell line selection and exclusive 3D organoid models. Benefit from OrganoidXplore™ and OmniScreen™ for rapid, in-depth analysis.

Learn More

In Vivo

Enhance drug development with our validated in vivo models, in vitro/ex vivo assays, and in silico modeling. Tailored solutions to optimize your candidates.

Learn More

Tissue

Experience ISO-certified biobanking quality. Access top biospecimens from a global clinical network, annotated by experts for precise research.

Learn More

Biomarkers and Bioanalysis

Leverage our global labs and 150+ scientists for fast, tailored project execution. Benefit from our expertise, cutting-edge tech, and validated workflows for reliable data outcomes.

Learn More

Data Science and Bioinformatics

Harness your data and discover biomarkers with our top bioinformatics expertise. Maximize data value and gain critical insights to accelerate drug discovery and elevate projects.

Learn More

KRAS

Accelerate innovative cancer treatments with our advanced models and precise drug screening for KRAS mutations, efficiently turning insights into clinical breakthroughs.

Learn More

EGFR

Advance translational pharmacology with our diverse pre-clinical models, robust assays, and data science-driven biomarker analysis, multi-omics, and spatial biology.

Learn More

Drug Resistance

Our suite integrates preclinical solutions, bioanalytical read-outs, and multi-omics to uncover drug resistance markers and expedite discovery with our unique four-step strategy.

Learn More

Patient Tissue

Enhance treatments with our human tumor and mouse models, including xenografts and organoids, for accurate cancer biology representation.

Learn More

Bioinformatics

Apply the most appropriate in silico framework to your pharmacology data or historical datasets to elevate your study design and analysis, and to improve your chances of clinical success.

Learn More

Biomarker Analysis

Integrate advanced statistics into your drug development projects to gain significant biological insight into your therapeutic candidate, with our expert team of bioinformaticians.

Learn More

CRISPR/Cas9

Accelerate your discoveries with our reliable CRISPR solutions. Our global CRISPR licenses cover an integrated drug discovery platform for in vitro and in vivo efficacy studies.

Learn More

Genomics

Rely on our experienced genomics services to deliver high quality, interpretable results using highly sensitive PCR-based, real-time PCR, and NGS technologies and advanced data analytics.

Learn More

In Vitro High Content Imaging

Gain more insights into tumor growth and disease progression by leveraging our 2D and 3D fluorescence optical imaging.

Learn More

Mass Spectrometry-based Proteomics

Next-generation ion mobility mass spectrometry (MS)-based proteomics services available globally to help meet your study needs.

Learn More

Ex Vivo Patient Tissue

Gain better insight into the phenotypic response of your therapeutic candidate in organoids and ex vivo patient tissue.

Learn More

Spatial Multi-Omics Analysis

Certified CRO services with NanoString GeoMx Digital Spatial Profiling.

Learn More

Biomarker Discovery

De-risk your drug development with early identification of candidate biomarkers and utilize our biomarker discovery services to optimize clinical trial design.

Learn More

DMPK Services

Rapidly evaluate your molecule’s pharmaceutical and safety properties with our in vivo drug metabolism and pharmacokinetic (DMPK) services to select the most robust drug formulations.

Learn More

Efficacy Testing

Explore how the novel HuGEMM™ and HuCELL™ platforms can assess the efficacy of your molecule and accelerate your immuno-oncology drug discovery programs.

Learn More

Laboratory Services

Employ cutting-edge multi-omics methods to obtain accurate and comprehensive data for optimal data-based decisions.

Learn More

Pharmacology & Bioanalytical Services

Leverage our suite of structural biology services including, recombinant protein expression and protein crystallography, and target validation services including RNAi.

Learn More

Screens

Find the most appropriate screen to accelerate your drug development: discover in vivo screens with MuScreen™ and in vitro cell line screening with OmniScreen™.

Learn More

Toxicology

Carry out safety pharmacology studies as standalone assessments or embedded within our overall toxicological profiling to assess cardiovascular, metabolic and renal/urinary systems.

Learn More

Our Company

Global CRO in California, USA offering preclinical and translational oncology platforms with high-quality in vivo, in vitro, and ex vivo models.

Learn More

Our Purpose

Learn more about the impact we make through our scientific talent, high-quality standards, and innovation.

Learn More

Our Responsibility

We build a sustainable future by supporting employee growth, fostering leadership, and exceeding customer needs. Our values focus on innovation, social responsibility, and community well-being.

Learn More

Meet Our Leadership Team

We build a sustainable future by fostering leadership, employee growth, and exceeding customer needs with innovation and social responsibility.

Learn More

Scientific Advisory Board

Our Scientific Advisory Board of experts shapes our strategy and ensures top scientific standards in research and development.

Learn More

News & Events

Stay updated with Crown Bioscience's latest news, achievements, and announcements. Check our schedule for upcoming events and plan your visit.

Learn More

Career Opportunities

Join us for a fast-paced career addressing life science needs with innovative technologies. Thrive in a respectful, growth-focused environment.

Learn More

Scientific Publications

Access our latest scientific research and peer-reviewed articles. Discover cutting-edge findings and insights driving innovation and excellence in bioscience.

Learn More

Resources

Discover valuable insights and curated materials to support your R&D efforts. Explore the latest trends, innovations, and expertly curated content in bioscience.

Learn More

Blogs

Explore our blogs for the latest insights, research breakthroughs, and industry trends. Stay educated with expert perspectives and in-depth articles driving innovation in bioscience.

Learn More

  • Platforms
  • Target Solutions
  • Technologies
  • Service Types

Leveraging Targeted Mouse I/O RNA-Seq Panels to Improve the Translatability of Immuno-Oncology Studies

In this post, we explore how a targeted RNA-Sequencing (RNA-Seq) panel can be incorporated into preclinical drug development to evaluate the impact of immune effectors in mediating anti-tumor immunity of novel immuno-oncology (I/O) agents. A case study highlights how RNA-Seq panels can complement traditional protein-based assays to gain a truly comprehensive picture of novel immunotherapies.

Precision Medicine: The Promise and the Challenges

Checkpoint blockade and other forms of cancer immunotherapies can induce durable clinical responses for a growing number of cancer indications. Remarkably, such responses are being observed in advanced cancer types for which effective therapies were previously not available.

However, similar to other anti-cancer treatments, only a subset of patients benefits from therapies that enhance anti-tumor immunity. This has led to an urgent need to identify novel biomarkers that can identify patients most likely to respond to immunotherapy.

Such biomarkers should ideally involve features of the tumor microenvironment (TME) since the composition and density of immune cells in the TME is well known to profoundly influence tumor progression, and efficacy of anti-cancer therapies.

Assays that allow for the genomic profiling of the TME are playing an increasingly important role in improving current and novel immunotherapy regimens.

Assays for TME characterization

High-throughput RNA-Seq has revolutionized cancer genomics and represents a promising avenue for accessing the complex molecular features and interactions of the TME.

Until now, protein-based methods such as fluorescence-activated cell sorting (FACS), immunohistochemistry (IHC) and immunofluorescence (IF) have been considered the gold standards in estimating the immune cell content within a sample.

While these techniques can still provide valuable insights, these methods have technical limitations that prevent them from being more broadly applicable. For instance, a continuous challenge is the availability, or lack thereof, of high-quality validated antibodies targeting potential protein biomarkers.

Further, IHC and IF, and to a lesser degree FACS, are limited in the number of markers that can be examined simultaneously (See Table 1 for some common limitations associated with the different methods).

Table 1. Methods used for the quantitative analysis of the TME.

Method Limitations
FACS Large amount of material needed and limited number of markers
IHC / IF Not representative if the tumor is heterogeneous and limited number of markers
RNA-Seq Provides no information on cellular composition
sc-RNAseq Expensive and bias due to tissue dissociation

Next-generation RNA-Seq is a powerful high-throughput substitute or complementary method to protein-based technologies and provides an accurate measure of gene expression across the entire genome without requiring the pre-selection of specific targets.

Moreover, the capacity to obtain both expression quantification and nucleic acid sequence information from RNA-Seq can be harnessed to measure immunological signals that are often obtained through multiple, distinct technologies.

With progress in single-cell RNA sequencing (sc-RNAseq), the complex interplay of tumor cells with the TME can now be reconstructed with great precision. These benefits can reduce costs and allow researchers to meet or fast-track project deadlines.

The Need for a Murine Next-Generation Sequencing (NGS)-Based Assay as a Basic Tool for Preclinical Drug Development

The prognostic value of RNA-based molecular features describing the TME has been demonstrated in recent clinical trials, where gene expression signatures for tumor infiltrating immune cells have been generated for several malignancies.

But this type of evaluation does not have to be restricted to the clinical phase of drug development.

Instead, a better understanding of the role the TME plays in an immunotherapy’s mechanism-of-action (MoA) and efficacy should ideally be developed before moving to human clinical studies.

By using assays that provide insights into the TME earlier in the drug development process, and across a range of preclinical models suitable for I/O studies, we aim to improve patient stratification to enhance both efficacy and safety of a novel agent.

Mouse Immuno-oncology targeted RNA-Seq panel (known as Mouse I/O RNA-Seq Panels) are available to evaluate genes that are associated with tumor immunity. This enables rapid transcriptomic insights into key immune cell populations and I/O pathways and processes in the TME (Figure 1).

For instance, in a Mouse I/O RNA-Seq available panel that encompasses 1,080 genes, immune cell signatures, surface markers, transcriptomic immune cell-specific biomarkers, and key pathways at the interface of the tumor, TME, and immune response call all be interrogated.

Figure 1. Characteristics of the Mouse I/O RNA-Seq panel

Characteristics of the Mouse I/O RNA-Seq panel

A typical workflow for a mouse I/O RNA-Seq panel should feature a simple design and offer flexibility in the source and type of study samples that can be analyzed (Figure 2).

Having flexibility in the workflow is important so that samples (e.g., extracted RNA, frozen tissue, whole blood, immune cell pellets) generated through syngeneic and MuPrime studies, or from in-house studies, or externally generated samples, can seamlessly be integrated into the workflow.

Finally, the final output should include both raw and normalized data as well as a comprehensive report with multiple group/condition comparisons.

Mouse I/O RNA-Seq panels are also amenable for large scale screening studies (such as the MuScreen in vivo pharmacology platform).

By using a collection of validated syngeneic and tumor homograft models that cover a range of cancer types and immune profiles, large-scale screening studies allow researchers a unique opportunity to:

  • Gain deep insights into an agent’s MoA
  • Identify biomarkers that may predict response
  • Use relevant preclinical models with human relevant cancers

Figure 2. Features of the Mouse I/O RNA-Seq panel workflow.

Features of the Mouse I/O RNA-Seq panel workflow

RNA-Seq platforms have several advantages over other commonly used transcriptomic technologies (Table 2 shows a comparison of these two assays).

A comparison study of RNA-Seq with Nanostring (a variation of DNA-based microarray technology) showed the mouse I/O RNA-Seq panel had higher accuracy and sensitivity, and the ability to discriminate between different mouse strains and carried shorter turnaround times rendering it more cost effective.

Table 2. Platform Comparison: mouse I/O RNA-Seq panel vs microarray based I/O profiling

Mouse I/O RNA-Seq panels can complement and enhance fluorescence-activated cell sorting (FACS)-based I/O profiling

Assay Comparison
  Mouse I/O RNA-Seq Panel Microarray I/O Profiling
Technology Targeted deep NGS Target mRNA (cDNA)
hybridize with DNA probes
Target Molecules mRNAs mRNAs
Throughput High (1080 genes) High (hundreds to
thousands of genes,
depending on vendor)
Accuracy High Low-medium
Sensitivity on detecting
low expressing genes
High Low-medium
Mouse strain discrimination Yes No
Turnaround time 2-3 weeks 3-10 weeks
depending on vendor
Cost Low Medium

Mouse I/O RNA-Seq panels can complement and enhance fluorescence-activated cell sorting (FACS)-based I/O profiling

As described above, RNA-Seq overcomes some key limitations of protein-based methods for TME profiling. Along with differences in target molecules, employing a mouse I/O RNA-Seq panel has much higher throughput compared to FACS (up to 1080 targets versus ~30), increased accuracy, and is suitable for large scale sample screening.

In addition, mouse I/O RNA-Seq panels can be used as a discovery tool while FACS is best suited for hypothesis testing and validation.

While a recognized limitation for RNA-Seq panels is the slower turnaround time as compared to some protein-based methods, RNA-Seq platforms do deliver more comprehensive sample analyses with comparatively lower costs on a per sample basis (see Table 3 for RNA-Seq Panel benefits).

Further, it is worth noting that Mouse I/O RNA-Seq platforms do not necessarily replace protein-based profiling methods, but they should be viewed as complementary for obtaining the most complete picture of immune cell content and effector pathway activation within tumor samples. This power of this complementary approach is highlighted in the following case study.

Table 3. Summary of Mouse I/O RNA-Seq Panel Benefits

Mouse I/O RNA-Seq Panel Benefits
Comprehensive
  • Expression level of 1080 genes from a single sample
  • Wide coverage in tumors, TME, and immune response
Flexible Sample
Types
  • Extracted RNA
  • Multiple tissues: tumor, blood, immune cells et.
High-Throughput
  • Suitable for large-scale immunotherapy screening,
    as well as examing multiple tissues in paralle
High Sensitivity
High Accuracy
  • >3 times the sequencing deoth of RNA-Seq
  • Avoid biases on non-protein coding RNAs like mitocondrai RNAs
Easy Integration
  • As standalone service or streamline with preclinical in vitro studies
  • Complementary with FACS, IHC and WB
Cost-Effective
Fast Turnaround
  • Lower cost per sample
  • 3-week turnaround time

Case Study: Determining the Response to Immunotherapy Under Varied Drinking Water pHs

Many studies have demonstrated that the gut microbiome (GM) plays an important role in immunotherapy response in both animal models and humans. In vivo preclinical variables, such as diet, housing, bedding and the pH of drinking water (such as acidified drinking water commonly used in research facilities), are known to influence the GM.

However, the effects of these variables on immunotherapy response remains unclear. This study therefore investigated the effects of drinking water pH on anti-PD-1 efficacy in MC38 murine colon cancer models using targeted RNA-Seq and FACS as complementary data sources.

RNA-Seq analysis of MC38 tumors was consistent with FACS data, confirming that mice receiving acidified water had the lowest total lymphocyte infiltration (CD45+), T cells (CD3+, CD4+ and CD8+) and highest percentage of NK cells.

These results indicate that acidified water can modulate the response to immunotherapy by inducing an immunosuppressive TME (Figure 3).

Figure 3. Immune signature (RNA-Seq) and immune cell phenotyping (FACS) analysis of MC38
tumors following anti-PD-1 therapy and administration of drinking water with different pH levels.

Immune signature and immune cell phenotyping

Leveraging the expanded number of immune signatures covered by a mouse I/O RNA-Seq Panel, pathway analysis revealed that hypoxia, MAPK and TGF-β signalling were also impacted by the pH, and alkaline conditions promoted antigen presentation.

Figure 4. Immune signature analysis of MC38 tumors treated with anti-PD-1
therapy followed by administration of drinking water a different pH levels.

Immune signature analysis of MC38 tumors

In the efficacy portion of the study, MC38 and CT26.WT colon cancer models were assessed. Different pH levels of drinking water were found to have a more pronounced impact on anti-PD-1 efficacy for the MC38 model.

Specifically, anti-PD-1 treatment in this model showed the largest tumor growth inhibition (TGI) with alkaline water (TGI=74%), followed by pH neutral water (TGI=66%) and then acidified water (TGI=58%).

Figure 5. Responses to anti-PD-1 treatment with drinking water at different
pH levels of MC38 and CT26.WT murine colon cancer models.

Responses to anti-PD-1 treatment

In summary, pH neutral to alkaline water led to expansion of immune effector cells, restraining of tumor suppressive subsets and activation of signalling pathways that promote anti-tumor immunity.

Based on these data:

  • Alkaline water is recommended for preclinical I/O efficacy studies that employ syngeneic mouse models
  • Alkaline drinking water should also be provided when exploring therapies intended to enhance the gut microbiome
  • Acidified water should be avoided when conducting I/O studies as it potentially weakens the response to therapies by modulating the microbiome community

Conclusion

Leveraging RNA-Seq panels offers researchers a variety of unique benefits that complement traditional protein-based assays. Together, these assays can help pinpoint an MoA and identify predictive biomarkers that can possibly improve the translatability of a new immunotherapy agent.

By incorporating strategies early in development that can fully characterize the TME, researchers have the best opportunity to accelerate their immunotherapy studies and position them for clinical success.

For more information on our Mouse I/O RNA-Seq panel, view our latest on-demand webinar.


Related Posts