A Multimodal Framework to Assess Patient-Derived Colon Tumor Organoid&#8211;Immune Cell Co-cultures

Mahshid Mohammadi; Nityanand Srivastava; Muzaffer Bhat; Terence Li; Emily Nghiem; Dior Dedushi; Natalie Thielen; Winfried Edelmann; Chaoyuan Kuang

doi:10.3791/71204

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Method Article

A Multimodal Framework to Assess Patient-Derived Colon Tumor Organoid–Immune Cell Co-cultures

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DOI:

10.3791/71204

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September 3rd, 2026

Mahshid Mohammadi^*¹^,² , Nityanand Srivastava^*¹^,³ , Muzaffer Bhat¹^,² , Terence Li¹^,² , Emily Nghiem¹^,⁴ , Dior Dedushi¹^,² , Natalie Thielen¹^,² , Winfried Edelmann¹^,³ , Chaoyuan Kuang¹^,²

¹Montefiore Einstein Comprehensive Cancer Center, Albert Einstein College of Medicine, ²Department of Oncology and Molecular Pharmacology, Albert Einstein College of Medicine, ³Department of Cell Biology, Albert Einstein College of Medicine, ⁴Department of Surgery, Albert Einstein College of Medicine

^* These authors contributed equally

Summary

This protocol describes the establishment of patient-derived colon tumor organoids in co-culture with autologous lymphocytes, followed by treatment with therapeutic small molecules and immune checkpoint inhibitors to evaluate T cell reactivity and treatment efficacy.

Abstract

T cells play a key role in cancer immunotherapy, and understanding their interactions with tumors is essential for developing novel immunotherapies. This protocol describes the step-by-step workflow for establishing colorectal cancer patient-derived organoids (PDOs) from our biobank and developing autologous PDO-T cell co-culture systems. Using Annexin V NIR and cleaved caspase-3 staining, we standardized flow cytometry protocols for surface and intracellular staining to assess T cell reactivity and cytotoxicity, as well as tumor organoid killing. In addition to flow cytometry-based analysis, the protocol includes methodological approaches for functional assays, such as ELISpot assays to quantify granzyme B and perforin secretion, and live-cell imaging to monitor T cell–mediated tumor killing over time via Annexin V–based apoptosis detection. This workflow further details the setup of co-culture conditions, including the incorporation of target inhibitors and immune checkpoint inhibitors in both monotherapy and combination treatment settings. Guidelines are provided for selecting and applying key immunological and tumor-associated markers, including CD137 for T cell reactivity, CD107a for T cell degranulation, caspase-3 for tumor apoptosis, and Ki67 for tumor proliferation. Collectively, this paper provides a comprehensive and reproducible framework for colorectal cancer organoid establishment, autologous T cell culture and expansion, co-culture setup, therapeutic perturbations, and downstream analyses using flow cytometry, ELISpot, confocal microscopy, and live-cell imaging.

Introduction

Colorectal cancer (CRC) remains one of the leading causes of cancer-related mortality worldwide despite advances in early detection and multimodal therapies¹. A major challenge in improving patient outcomes is the lack of preclinical models that accurately recapitulate the complex architecture, intratumoral heterogeneity, tumor–immune interactions, and therapeutic responses observed in human CRC²^,³^,⁴^,⁵^,⁶. Conventional models, including genetically engineered mouse models (GEMMs....