Metastasis Models
In Vivo Pharmacology

Metastasis Models

In vivo metastasis models are designed to analyze the anti-metastatic effect of novel anti-cancer drug candidates.

Metastasis mouse models comprise two types: 1. Orthotopic: Metastatic cells originate from the orthotopically growing tumors, followed by the migration of tumor cells to the metastasis site. 2. Disseminated: Heart or tail vein injection of tumor cells result in the establishment of metastases in the lung, bone marrow, or brain depending on the metastasis model’s tumor cell line and route of administration. The various metastasis models enable differential examination of the metastasis process.

Reaction Biology offers syngeneic and xenograft metastasis models with advanced imaging options for whole-body monitoring of metastasis.

  • Whole-body imaging via IVIS allows monitoring of tumor growth kinetics and therapeutic efficacy of a drug candidate
  • Models are established with different injection routes to mimic different metastasis patterns
  • In vivo metastasis model study reports are written by PhD-level medical writers and are custom-tailored for each project
  • Meticulous documentation in line with ISO 9001 certification and GV-Solas requirements

Reaction Biology has a team of global business development managers free to discuss in most time zones. Contact us today for inquiries about our xenograft and syngeneic metastasis models or to directly request a quote.

Metastasis Patterns

Reaction Biology’s in vivo metastasis models allow the differential examination of metastasis location. A. Orthotopic breast tumor cells migrate to the lungs. B. Tumor cells disseminate into different parts of the body such as bone marrow or lungs after intravenous injection. C. Tumor cells injected intracardially on the other hand settle in the brain.

Metastasis Options

Metastasis Options

Browse our metastasis models

In Vivo Metastasis Studies

  • Syngeneic Study Example
  • Xenograft Study Example
  • Study Layout
  • Study Options
  • Imaging
  • Custom Model Development
  • Certification
  • Quality Assurance
  • Animal Welfare
Syngeneic Study Example

Goal: A standard of care drug was tested for its potential to inhibit metastasis from orthotopically growing breast tumors 4T1.

Cell line: For 4T1 metastasis studies we use the cell line 4T1-Luc-M3. The parental 4T1 cell line was transduced to stably express firefly luciferase enabling detection of metastasis via luciferase quantification. Moreover, we have generated the 4T1 cell line M3 which shows a very homogeneous metastasis pattern. The M3 cell line was generated by isolation of lung metastasis that originated in orthotopically growing 4T1 tumors. We reimplanted the lung metastasis into the mammary fat pad and again isolated developing lung metastasis. We performed this cycle of subpopulating three times. The cell lines were subsequently tested via STR profiling to determine their 4T1 origin.

Study layout: 4T1-Luc-M3 cells were implanted into the mammary fat pad of Balb/C mice, at 12 mice per group. After randomization on day 4, treatment was initiated. The test compound was administered intravenously at 12 mg/kg on days 4, 9, and 16 after implantation. Primary tumor growth was monitored via caliper twice weekly, animal weight was measured three times per week, animal behavior was observed daily. On day 21, animals were euthanized and endpoint measurements for the determination of primary tumor volumes, and wet weights were performed with primary breast tumors. Selected organs (lymph nodes (a mixture of inguinal/axillary and brachial), ileum, spleen, kidney, spine, femur, liver, heart, lung, and brain) were collected, homogenized, and subjected to enzymatic luciferase quantification to determine metastasis burdon.

4T1 metastasis model animal weight

Animal weights for the 4T1-Luc_M3 metastasis model are shown with vehicle and drug treatment after primary tumor implantation.

tumor growth of 4T1 metastasis model with treatment of doxorubicin

Monitoring of primary 4T1-Luc-M3 tumor growth was performed twice per week via caliper measurement. Shown are the mean values +/- SEM. The vehicle control group comprised 12 mice during the entire study duration. 4 mice of the treatment group died before the termination date on day 22.

4T1 metastasis distribution in the body and different organs

On day 21 after primary tumor implantation, both the vehicle control and the drug treatment groups were euthanized. A selection of organs was isolated from the animals of the vehicle group and metastasis burdon was quantified via ex vivo enzymatic luciferase quantification. The insert shows the values for each mouse, the bar graph shows the mean +/- SEM for all vehicle control animals.
The lung showed the highest counts of luciferase-expressing metastasis. 11 out of 12 animals had detectable lung metastasis. The lymph nodes of almost all animals also show metastasis.

Xenograft Study Example

Goal: To validate the differences in the metastasis pattern for different routes of tumor cell injection, we have compared MDA-MB-231 metastasis in mice after tumor cell injects via intravenous and intracardiac routes.

Cell line: For this study, we have used the breast cancer cell line MDA-MB-231-Luc-Z1 which was transduced for stable expression of firefly luciferase to enable live imaging of tumor cells in the mouse body via bioluminescence measurement.

Study layout: MDA-MB-231 breast tumor cells were implanted via intravenous route into the tail vein and via intracardiac route into the left ventricle of BALB/c nude mice. Metastasis growth was monitored via whole-body bioluminescence imaging once per week, animal weights were taken three times per week and animal behavior was monitored daily. At days 32 and 37, respectively, the mice were euthanized and selected organs were isolated and homogenized before enzymatic quantification of luciferase which serves as a readout for metastasis load.


animal weight monitoring in metastasis study for comparison of intravenous and intracardiac

Animal weights were measured after i.v. (green) and intracardiac (purple) injection of MDA-MB-231 tumor cells into mice.


metastasis growth after intreavenous or intracardiac implantation


The proliferation of tumor cells in the mice was determined via whole-body bioluminescence measurement. Data are displayed as mean +/- SEM.


metastasis load in various organs after implantation intravenous or intracardiac

Metastasis load is shown after i.v. (green) and intracardiac (purple) injection for a variety of organs at the endpoint of the study on day 32 and day 37, respectively. The organs were homogenized and subjected to the ex vivo luciferase assay. Shown are individual counts of metastasis load for each mouse and the corresponding mean values. LN ing…inguinal lymph node; LN ax/br … axillary lymph node.


Result: MDA-MB-231 cells proliferate with similar rates in BALB/c nude mice after implantation into the tail vein or via the heart. Tumor cells implanted into the tail vein are transported to the heart and circulate via the pulmonary artery into the lung capillaries. The high count in the lungs suggests that most cells are filtered in the lungs from the blood circulations. Tumor cells implanted into the left ventricle of the heart leave via the aorta to enter the systemic circulation which shows in a high count of metastasis in the brain and other organs.

Study Layout

Standard study layout

Luciferase-expressing tumor cells are implanted into the mice (orthotopic, intravenous, intracardiac, bone-marrow engraftment). When appropriate luciferase signals are detected by whole-body luminescence measurement, animals are randomized in treatment groups before treatment starts. The mice are monitored on a regular basis: metastasis is measured via whole-body luminescence measurement once weekly, animal weights are taken three times per week and animal behavior is observed daily. Once per week, we update the customer with a graphical presentation of the study progress.


A comprehensive report will be prepared by a PhD-level medical writer. The report will be custom-tailored for each project with data that can be used for filing official documents. The report includes material, methods, raw data, animal health chart, and graphs plus statistical evaluation.

Selection of optional services:

  • Isolation of tumors at necropsy for determination of volume and wet weight
  • Preservation of tissue via fixation or snap-freezing
  • Histological or pathological analysis of tumor tissue and mouse body
  • Determination of expression of genes or proteins
  • Plasma sampling and bioanalysis of compound or metabolites
  • Quantification of cytokines and chemokines in blood or tissue via MESO QuickPlex SQ120 multiplex analysis
  • Flow cytometry analysis for immune cell profiling
  • MRI analysis via collaboration
  • Whole-body imaging to detect metastatic foci
  • Ex vivo luciferase measurement of organs to determine the metastatic load
Study Options

Standard Efficacy Studies: The therapeutic effect of a new drug is tested by quantification of metastatic load in the whole body and a selection of organs.

Proof-of-Concept Studies: New drug candidates need to be tested not only for efficacy but also for their mechanism of action in the animal. Exploratory proof of concept studies can be performed together with histological, pathological, or molecular investigations of the biological activity of the drug on the tumor tissue.

Pharmacokinetic Studies: Pharmacokinetic studies on tumor-bearing mice are helpful in determining the concentration of drugs not just in plasma but in the tissue of the tumors.

Pharmacodynamic Studies: Pharmacodynamic studies elucidate whether and how a drug acts on its target.

Dose-Response-Relationship: Determine suitable drug doses for efficacy testing.

Drug Combination Studies: Combinations of drugs can lead to synergistic effects and vastly increased tumor response.

Survival Studies: A survival study gives data in addition to the reduction of tumor growth – it shows the number of partial and complete responders. The setup is also useful for drugs with no homogeneous response. Studies with an outcome of a mix of non-responders and responders are hard to interpret with common statistical values due to the high standard variation of the results. Survival studies can give meaningful results for such ambivalent drug candidates.


IVIS Spectrum In Vivo Imaging System by PerkinElmer.

Using the IVIS system, we can detect and track bioluminescent and fluorescent reporters across the blue to near-infrared wavelength region. This state-of-the-art system is equipped with 10 excitation filters for detection of multiple signals in one mouse.

Custom Model Development

We continuously increase our portfolio of tumor models and provide custom development of new tumor models.

Cell lines: We can develop new tumor models a. based on customer-provided cell lines, b. based on a cell line from our extensive database of almost 200 cell lines or c. purchase a cell line from a commercial vendor.

Two-step approach: In case no tumor-model related data are available, we will perform a two-step approach to develop a new tumor model. In the first step, we identify a suitable mouse strain for tumor growth and determine the optimal conditions for tumor cell implantation. In a second study, we will monitor the tumor growth after tumor cell implantation into 12 mice for the determination of the growth characteristics of the tumors. This two-step approach guarantees satisfactory study results for subsequent efficacy studies.

Standard of care (SOC) treatment: Most of our models were tested with a variety of standard of care treatment. Please let us know which positive control is the most suitable for your drug candidate, and we will be happy to provide historical data with SOC treatment options.


The animal facility of Reaction Biology is located in Freiburg, Germany.

Our facility is certified under ISO 9001:2015, which is an international standard that specifies requirements for the quality management system and demonstrates the ability to consistently provide products and services that meet customer and regulatory requirements. Reaction Biology is committed to continuously maintain and improve its quality management system as a key element for the achievement of the highest customer satisfaction. ISO 9001
Quality Assurance

Animal work

  • Routine health monitoring of sentinel animals (according to FELASA guidelines)
  • Standardized operation procedures are in place of every step and every model

Cell lines

  • Routine authentication of tumor cell lines by STR profiling
  • Mycoplasma testing of tumor cells by PCR just prior to implantation

Study support

  • Studys generally start (tumor implantation) 3 to 5 weeks after receipt of order
  • Suggestions for study layout to get statistically relevant results
  • Weekly study update and personal contact with study supervisor
  • Reports are written by technical writers on PhD level

Ethical principles

  • Our animal work is conducted according to the 3R (Replacement, Reduction, and Refinement)
  • We are working closely with our animal care committee to ensure timely adaptions of our animal care licenses to custom-tailor our client’s project to ensure the most meaningful study outcome
Animal Welfare

Reaction Biology uses laboratory animals to help our customers to understand the fundamental mechanisms behind malignancies and to discover therapeutics to prevent and treat cancer. Data obtained from animal models is critical in predicting the clinical outcome for an oncology drug candidate in development.

Animal welfare is of the utmost importance to us. Animal-based research is highly regulated to ensure ethical and responsible treatment. The mice in our facility are specifically bred for research purposes, and they are cared for to the very high standards.

We are working under GV-SOLAS and ISO9001 regulations in regard to standards of animal welfare and code of practice.

We employ three veterinarians and appropriately trained staff to ensure animal welfare is maintained at the highest standards. Regulation officers inspect our unit regularly.