1

Selection of Immunodeficient Mouse Strains

Several mouse strains are commonly used for PDX development, including:

• Athymic nude mice
• Severe Combined Immunodeficient (SCID) mice
• Non-Obese Diabetic SCID (NOD-SCID) mice
• Rag2 knockout mice

NOD-SCID mice are frequently preferred over conventional SCID mice due to their lack of functional natural killer (NK) cells, resulting in improved tumor engraftment rates. Currently, NSG mice (NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ) are widely regarded as the most efficient strain for PDX establishment. These mice carry a null mutation in the interleukin-2 receptor gamma chain (IL2Rγ), leading to defective cytokine signaling and profound impairment of innate immunity, including absence of NK cell activity. This severe immunodeficiency supports highly efficient engraftment of primary human tumors.

2

Source Material for PDX Generation

Two primary types of tumor input material can be used:

1. Tumor tissue fragments
2. Single-cell suspensions

Tumor specimens are typically collected during surgical resection and transported in sterile culture medium such as DMEM or RPMI supplemented with fetal bovine serum and antibiotics. Upon arrival in the laboratory, necrotic areas are removed.

Tumor Fragment Approach

Tumors are sectioned into small fragments (approximately 2–3 mm³) for implantation. This method preserves:

• Cell–cell interactions
• Extracellular matrix components
• Partial tissue architecture
• Microenvironmental characteristics

As a result, tumor fragments more accurately recapitulate the original tumor biology.

Single-Cell Suspension Approach

Alternatively, tumor tissue may be mechanically or enzymatically dissociated into a single-cell suspension. This strategy allows:

• More uniform sampling of the entire tumor
• Reduced spatial bias in clonal representation

However, enzymatic digestion and mechanical disruption can reduce cell viability and increase sensitivity to anoikis, potentially lowering engraftment success.

3

Implantation Procedures

Unlike xenograft models generated from cultured cell lines, PDX development does not require intermediate in vitro expansion. Tumor fragments can be implanted directly after processing, avoiding prolonged culture steps that may alter tumor biology.

Implantation can be performed using two main approaches:

Heterotopic Implantation

Tumor fragments are implanted into anatomical sites unrelated to the tumor’s origin, most commonly:

• Subcutaneous flank region
• Subrenal capsule

Subcutaneous models are technically simple and allow easy tumor monitoring but rarely develop metastases and do not fully replicate the native tumor microenvironment.

Orthotopic Implantation

Tumor tissue is implanted into the corresponding organ of origin. Although technically more demanding, orthotopic models better reproduce:

• Native tissue microenvironment
• Local invasion
• Metastatic potential

Orthotopic PDX models are generally considered more biologically relevant.

4

Engraftment and Latency

Tumor engraftment typically requires 2 to 4 months, depending on:

• Tumor type and aggressiveness
• Implantation site
• Mouse strain used

Failure of engraftment should not be concluded until at least six months without tumor growth.

5

Passaging and Expansion

The first recipient mice are designated as the F0 generation. Once tumors reach an appropriate size, they are harvested and re-implanted into new recipient mice, generating subsequent passages (F1, F2, F3, etc.).

For drug development studies, PDX models beyond the F3 generation are commonly used, provided that molecular and histological fidelity to the original patient tumor has been confirmed.

6

Cryopreservation and Biobanking

After tumor harvest:

• Tissue is sectioned into 1–2 mm³ fragments
• Washed multiple times in culture medium
• Placed in freezing medium (FBS with 10% dimethyl sulfoxide)
• Stored at −80°C for long-term preservation

For re-implantation, frozen tumor stocks are rapidly thawed in a 37°C water bath, washed in culture medium containing antibiotics, and prepared for transplantation into recipient mice.