Lentiviral Organoid Engineering: Overexpression to CRISPR Screens

In the previous two articles, we introduced the fundamentals of lentiviral vectors and organoid models, as well as practical strategies for improving lentiviral transduction efficiency in organoids.

In this article, we focus on real-world research applications. These examples illustrate how lentiviral transduction of organoids can be used in laboratory studies and how researchers can select an appropriate viral vector strategy for different experimental objectives.

1. Classic Applications of Lentiviral Vectors in Organoids

1.1 Engineering Genetically Modified Prostate Cancer Organoid Models

To model the complex genetic alterations and lineage transitions that occur during prostate cancer progression, researchers developed genetically engineered tumor models based on prostate cancer organoids.

Using gene-editing approaches, the researchers generated organoids with different tumor suppressor gene deficiencies and used lentiviral vectors to stably express oncogenes in prostate organoids, creating organoid models carrying different combinations of cancer driver genes.

The genetically engineered organoids were subsequently used for in vivo transplantation and tumor progression studies. This model provided a platform for investigating the molecular mechanisms underlying the transition of prostate cancer toward a neuroendocrine phenotype and for identifying key genetic drivers of tumor progression.

Figure 1. Genetic engineering of prostate cancer organoids using lentiviral vectors. [PMID: 39394434]

1.2 Establishing a Colorectal Cancer Organoid Model for Tumor Immunology

To investigate mechanisms of immune evasion in colorectal cancer with a low mutational burden, researchers established an engineered tumor model based on colorectal cancer organoids.

The study used lentiviral vectors to stably deliver Apc knockdown and a neoantigen expression cassette into mouse colon organoids, generating colorectal cancer organoids with sustained antigen expression.

The engineered organoids were subsequently combined with gene-editing approaches and mouse transplantation models to investigate how neoantigen expression levels affect tumor immune recognition and therapeutic responses.

This study demonstrates the potential of lentiviral transduction for stable genetic modification of organoids and for developing organoid-based tumor immunology models.

Figure 2. Establishment of a colorectal cancer organoid model for tumor immunology studies. [PMID: 34738089]

1.3 Lentiviral CRISPR Screening in Organoids to Study Drug Response

To investigate the mechanisms regulating drug sensitivity in gastric cancer, researchers established a CRISPR screening platform based on human gastric cancer organoids.

The study used lentiviral vectors to achieve stable Cas9 expression in gastric cancer organoids, generating organoid models with efficient gene-editing capabilities.

The researchers subsequently combined CRISPR knockout screens with single-cell transcriptomic analysis to systematically identify regulatory genes associated with cisplatin response.

This work demonstrates the value of lentiviral vectors for stable genetic engineering and functional screening in organoid models, providing an additional experimental framework for investigating drug-response mechanisms using human-derived organoids.

Figure 3. Establishment of a CRISPR screening platform using gastric cancer organoids. [PMID: 40813572]

2. Customer Case Studies

The studies above highlight the broad potential of lentiviral vectors in organoid-based research. In practical research projects, this combination has also been applied to functional validation across multiple cancer types.

Below are three selected research cases representing three different applications: gene overexpression, gene knockdown, and pathway regulation.

2.1 APOA1 Overexpression in Gastric Cancer Organoids and Immunotherapy Resistance

A research team at Sun Yat-sen University Cancer Center used multi-omics analysis from the NEOSUMMIT-01 clinical trial to investigate the genetic and immune determinants underlying heterogeneous responses to neoadjuvant immunochemotherapy (nICT) in gastric cancer.

The researchers identified five tumor microenvironment ecotypes (EC1–EC5). Among them, EC5, characterized by enrichment of immunosuppressive macrophages, showed the poorest response to nICT.

The underlying resistance mechanism was associated with interactions between APOA1-positive tumor cells and TREM2-positive macrophages mediated by lipoprotein secretion.

To functionally validate this mechanism, researchers used lentiviral vectors to overexpress Apoa1 in mouse gastric cancer organoids (mGCOs) and subsequently transplanted the engineered organoids into mice.

Apoa1 overexpression significantly promoted tumor growth. Flow cytometry further demonstrated increased infiltration of TREM2-positive macrophages and activation of the PI3K and SYK signaling pathways.

These findings provided functional evidence that APOA1-positive tumor cells may drive immunotherapy resistance by remodeling macrophage function. The study also provided a potential rationale for targeting the APOA1–TREM2 axis to overcome nICT resistance and may inform patient stratification and combination treatment strategies in future clinical trials.

Figure 4. Lentiviral-mediated gene overexpression in mouse gastric cancer organoids. [PMID: 41720086]

2.2 RBM4 Knockdown in Organoids Reveals Its Tumor-Promoting Role in Ovarian Clear Cell Carcinoma

A research team at Peking Union Medical College Hospital used Ribo-seq combined with RNA-seq to investigate the role of RBM4 in ovarian clear cell carcinoma (OCCC).

Although RBM4 mRNA levels were reduced in OCCC, its translational efficiency was significantly increased, resulting in elevated protein expression.

To investigate the biological significance of this translational regulation, researchers used lentiviral vectors to achieve stable RBM4 knockdown in both OCCC cell lines and patient-derived organoids.

RBM4 knockdown resulted in reduced organoid size and viability, accompanied by a significant decrease in intracellular glycogen accumulation.

These findings provided functional evidence in a 3D organoid model that RBM4 promotes OCCC progression through regulation of glycogen metabolism, supporting its potential as a therapeutic target in ovarian clear cell carcinoma.

Figure 5. Lentiviral-mediated gene knockdown in human OCCC organoids. [PMID: 42249082]

2.3 USP20 Knockout Reveals Its Role in Metabolic Reprogramming and Stromal Activation in Pancreatic Cancer

A research team at Lanzhou University Second Hospital investigated how inflammatory signaling promotes pancreatic cancer progression and stromal activation through the STAT3–USP20–HMGCR axis.

The study found that this signaling pathway can reprogram cholesterol metabolism and contribute to malignant progression and stromal activation in pancreatic cancer.

To functionally investigate the role of USP20, researchers used lentiviral vectors to knock out USP20 in pancreatic cancer organoids.

USP20 loss significantly inhibited organoid proliferation, suppressed YAP/TAZ signaling, and reduced cancer-associated fibroblast (CAF) activation.

The researchers further used AAV-mediated in vivo knockdown in a KPC pancreatic cancer model. USP20 deficiency inhibited tumor growth, reduced stromal deposition, and enhanced the efficacy of anti-PD-1/CTLA-4 immunotherapy.

Together, these findings provide functional evidence supporting the USP20 metabolic–stromal regulatory axis as a potential target for improving immunotherapy responses in pancreatic cancer.

Figure 6. Lentiviral-mediated gene knockout in pancreatic cancer organoids. [PMID: 41196022]

3. Choosing the Right Lentiviral Strategy for Organoid Research

These studies demonstrate that lentiviral vectors are no longer used simply as tools for gene delivery. They have increasingly become an important platform for organoid engineering, disease modeling, mechanistic studies, and functional screening.

Different research objectives require different genetic manipulation strategies:

Therefore, when designing an organoid gene delivery experiment, researchers should consider the biological question, desired genetic manipulation, organoid type, transduction efficiency, and downstream analysis requirements when selecting the appropriate lentiviral strategy.

4. OBiO Tech: Lentiviral Solutions for Organoid Research

To support researchers facing the challenges of lentiviral transduction in organoid models, OBiO Tech provides an integrated workflow covering lentiviral vector construction and viral packaging.

Our proprietary Vpack high-titer packaging system enables lentiviral titers of 10⁸–10⁹ TU/mL, supporting organoid models with different requirements for viral input and transduction efficiency.

Depending on the target organoid and experimental objective, researchers can select lentiviral vectors with different envelope types to improve compatibility with different cell sources and organoid models.

OBiO Tech's service covers the complete workflow from vector construction to lentiviral packaging, providing an integrated solution for organoid gene delivery and genetic engineering.

For additional technical parameters and experimental condition references, researchers can contact the OBiO Tech technical support team for tailored guidance.