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- Spatial Profiling Uncovers Immune Correlates of Durable Response in Phase 1/2 MSS Colorectal Cancer Trial

Research Spotlight

# Spatial Profiling Uncovers Immune Correlates of Durable Response in Phase 1/2 MSS Colorectal Cancer Trial

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Reference: Christenson, E.S., Wala, J.A., Parkinson, R. et al. Copanlisib in combination with nivolumab for microsatellite stable colorectal cancer: a phase 1/2 trial. Nat Commun 17 , 7114 (2026). [https://doi.org/10.1038/s41467-026-72985-6](https://doi.org/10.1038/s41467-026-72985-6)

## Clinical Context & Translational Challenge

Evaluating novel immunotherapies in microsatellite stable (MSS) colorectal cancer (CRC), a disease representing ~95% of metastatic cases and historically non-responsive to single-agent checkpoint blockade, requires resolving complex spatial architecture from limited clinical specimens. In an investigator-initiated Phase 1/2 trial evaluating copanlisib (pan-PI3K inhibitor) plus nivolumab (anti-PD-1) in 39 heavily pretreated patients, a molecularly defined subgroup harboring PI3K alterations achieved remarkable clinical benefit. All partial responses occurred within this subgroup, yielding durable progression-free survival spanning 24.2 to over 33.7 months. However, extracting deep spatial insights from small metastatic core biopsies without exhausting finite tissue blocks remains a major challenge in clinical trials.

## High-Plex Tissue Profiling via Orion®: Deep Biomarker Data from a Single Section

To overcome the tissue constraints inherent to metastatic core biopsies, trial investigators performed Orion multiplex immunofluorescence spatial profiling on 38 total pre- and on-treatment core biopsies across 20 patients, with 18 pre-treatment and 8 paired on-treatment biopsies meeting full quality control criteria for single-cell spatial profiling. Orion enabled rapid, single-pass fluorescence imaging of an 18-marker immune-focused panel at subcellular resolution from a single 5 µm FFPE section per biopsy. By capturing comprehensive cell phenotypes and spatial relationships on one slide, the platform eliminated the need for multi-section cutting or harsh cyclic processing, conserving precious patient biopsy tissue for parallel genomic and proteomic analyses. Furthermore, quantitative T-cell infiltration measurements generated by Orion on single sections demonstrated high analytical concordance when cross-validated against serial sections profiled via t-CyCIF on a RareCyte CyteFinder® platform.

## Linking Peripheral Immunity to Tumor Microenvironment Dynamics

Spatial analysis of baseline biopsies ( n = 18) using Orion identified clear microenvironmental predictors of therapeutic response. Clinical responders exhibited significantly higher pre-treatment intratumoral CD3 + CD8 + T-cell density and an elevated CD8 + /FOXP3 + Treg ratio. While PIK3CA mut tumors showed overall enrichment for CD163 + macrophages, patients exhibiting a higher ratio of macrophage-to-tumor PD-L1 expression were significantly more likely to achieve a partial response.

Importantly, Orion's spatial profiling provided an in-situ counterpart to liquid suspension cytometry, bridging systemic immune monitoring with intact tissue biology. While mass cytometry (34-plex CyTOF) on peripheral blood mononuclear cells (PBMCs) identified an on-treatment expansion of circulating activated effector CD8+ T cells, Orion imaging directly corroborated this systemic response within tissue, capturing an increase in intratumoral CD3+CD8+ T-cell infiltration in 100% of evaluable paired biopsies (8 out of 8 patients at 6 weeks, Cycle 2 Day 15). However, while liquid cytometry confirmed peripheral activation, Orion’s spatial resolution was essential to show that immune cell recruitment into the tumor is insufficient on its own, as localized, tumor-intrinsic signaling (such as compensatory ERK/MAPK upregulation) continues to drive resistance.

## Conclusion

The combination of copanlisib and nivolumab led to durable responses in patients with MSS metastatic CRC with alterations in the PI3K pathway. This treatment regimen was expected to remodel the immune landscape and perturb tumor signaling pathways, and Orion spatial proteomic analysis was used to capture these changes. Orion’s single-round high-plex approach provides the biomarker depth, large cohort capacity, and data quality necessary to explain precision therapy-induced changes in the tumor microenvironment. These critical biologic insights are expected to guide future trial design and to provide predictive biomarkers of clinical response to inform which patients are most likely to benefit from the treatment approach.