Development of cutting-edge preclinical models to study the response of small-cell lung cancer to current therapies

Abstract

Introduction

Small cell lung cancer (SCLC) is the most aggressive subtype of lung cancer. Although the combination of chemotherapy and immune checkpoint inhibitors has recently shown slight improvement in outcome, the prognosis remains very poor. The development of relevant preclinical models is a crucial step towards new therapies. Here, we present the models we are currently using and developing in the laboratory: xenografts derived from Circulating Tumor Cells from SCLC patients (CDXs), CDXs on humanized mice (Hu-CD₃₄₊-NXG), and CDXs on a cutting-edge combination of Hu-CD₃₄₊-NXG with controlled gut microbiota (‘MESHCAP’ model, meaning Exogenous Microbiota and Humanized Mice for Lung Cancer).

Methods

CTCs are collected from patients blood at the time of SCLC diagnosis. Isolation is performed by immunomagnetic negative selection, followed by a flow cytometry sorting of CD₅₆₊/CD₄₅₋ cells labeled with fluorescent antibodies.

For CDXs generation, CTCs are injected subcutaneously into immunodeficient NSG mice.

For humanized CDXs, CDXs are subcutaneously grafted into humanized CD₃₄₊ NXG mice. To control gut microbiota composition (MESHCAP model), the original microbiota from mice is depleted by a specific cocktail of antibiotics. NXG mice are then inoculated with a specific bacterial consortium prior to CD₃₄₊ humanization.

In all three models, subcutaneous tumor growth is monitored manually. When tumor size reaches 1000 mm³, the animals are sacrificed and the tumor removed. Tissues are fixed and used for immunochemistry, or snap-frozen for subsequent genetic or transcriptomic analysis.

Results

We have developed a unique method for isolating live CTCs from the blood of SCLC patients. This EpCAM-independent method is based on the CD₅₆₊ marker frequently expressed on the membrane surface of SCLC cells.

To date, we have successfully generated 5 CDXs from CTCs of SCLC patients. These models show rapid tumor growth, supporting the tumorigenic properties of CD₅₆₊-CTCs. Initial trials of CDXs grafted onto humanized mice show a partial response to immunotherapy.

Development of our MESHCAP mouse model is underway, and preliminary results on depletion of gut microbiota and humanization of the immune system are encouraging. Our bacterial consortium, supposedly beneficial for immunotherapy, has been cultured and will be inoculated in mice.

Conclusion

We have developed CDX-based mouse models that are relevant and effective for studying response to SCLC treatment. While conventional CDXs models allow us to study response to chemotherapy, inhibitors etc., transplantation of humanized mice broaden research possibilities by enabling us to test immunotherapeutic strategies. We also expect to analyze the impact of gut microbiota composition on immunotherapy with our new MESHCAP mouse model.