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Updated: Nov 15, 2025
Microfluidic Co-Culture Models for Dissecting the Immune Response in in vitro Tumor Microenvironments
Published on: April 30, 2021
Multimodal pooled Perturb-CITE-seq screens in patient models define mechanisms of cancer immune evasion
Chris J Frangieh1,2, Johannes C Melms3,4, Pratiksha I Thakore1
1Klarman Cell Observatory, Broad Institute of MIT and Harvard, Cambridge, MA, USA.
Abstract:
Resistance to immune checkpoint inhibitors (ICIs) is a key challenge in cancer therapy. To elucidate underlying mechanisms, we developed Perturb-CITE-sequencing (Perturb-CITE-seq), enabling pooled clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 perturbations with single-cell transcriptome and protein readouts. In patient-derived melanoma cells and autologous tumor-infiltrating lymphocyte (TIL) co-cultures, we profiled transcriptomes and 20 proteins in ~218,000 cells under ~750 perturbations associated with cancer cell-intrinsic ICI resistance (ICR). We recover known mechanisms of resistance, including defects in the interferon-γ (IFN-γ)-JAK/STAT and antigen-presentation pathways in RNA, protein and perturbation space, and new ones, including loss/downregulation of CD58. Loss of CD58 conferred immune evasion in multiple co-culture models and was downregulated in tumors of melanoma patients with ICR. CD58 protein expression was not induced by IFN-γ signaling, and CD58 loss conferred immune evasion without compromising major histocompatibility complex (MHC) expression, suggesting that it acts orthogonally to known mechanisms of ICR. This work provides a framework for the deciphering of complex mechanisms by large-scale perturbation screens with multimodal, single-cell readouts, and discovers potentially clinically relevant mechanisms of immune evasion.
Insights
New Perturb-CITE-seq technology reveals CD58 loss as a novel mechanism of immune evasion in melanoma, contributing to resistance against immune checkpoint inhibitors (ICIs). This discovery offers new avenues for cancer therapy.
Area of Science:
- Immunology
- Cancer Biology
- Genomics
Background:
- Resistance to immune checkpoint inhibitors (ICIs) poses a significant challenge in cancer treatment.
- Understanding the molecular mechanisms underlying this resistance is crucial for developing effective therapies.
Purpose of the Study:
- To develop and apply a novel Perturb-CITE-sequencing (Perturb-CITE-seq) platform to identify mechanisms of cancer cell-intrinsic ICI resistance (ICR).
- To uncover novel pathways and molecular players involved in immune evasion.
Main Methods:
- Developed Perturb-CITE-seq, integrating pooled CRISPR-Cas9 perturbations with single-cell transcriptome and protein profiling.
- Applied the platform to patient-derived melanoma cells and tumor-infiltrating lymphocyte (TIL) co-cultures, analyzing ~218,000 cells across ~750 perturbations.
- Profiled RNA and 20 proteins to assess functional impacts on ICI resistance.
Main Results:
- Re-identified known ICR mechanisms, including defects in interferon-gamma (IFN-γ)-JAK/STAT and antigen-presentation pathways.
- Discovered CD58 loss/downregulation as a novel mechanism conferring immune evasion.
- Observed CD58 downregulation in tumors from melanoma patients with ICR, independent of IFN-γ signaling and MHC expression.
Conclusions:
- Perturb-CITE-seq provides a powerful framework for dissecting complex biological mechanisms using multimodal single-cell readouts.
- CD58 represents a potentially clinically relevant target for overcoming ICI resistance in melanoma.
- CD58-mediated immune evasion acts orthogonally to established ICR mechanisms, highlighting a new therapeutic strategy.
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