Converting cold to hot: Strategies to sensitize microsatellite-stable colorectal cancer to immunotherapy

Table 1 Major immunobiological barriers to immune checkpoint inhibition in microsatellite-stable/proficient mismatch repair colorectal cancer and actionable therapeutic opportunities

Immune barrier/feature	Mechanistic basis	Representative readouts/contexts	Actionable strategies
Low immunogenicity and weak antigen presentation	Low mutation load (MSS/Low TMB); impaired APC priming; HLA/B2M loss; IFN-JAK/STAT defects	Low TMB; low IFN-γ/CXCL9-CXCL10; MHC-I downregulation	Chemo/RT priming; STING/TLR agonists; epigenetic priming; vaccines/neoantigen approaches[13-15,25]
T cell exclusion by stromal/TGF-β programs	CAF-derived TGF-β; dense ECM; reduced trafficking/retention immuneexcluded phenotype	CMS4/stromal signature; high TGF-β; desmoplasia	TGF-β blockade/traps; CAF/ECM modulation; CXCR4 blockade; vessel normalization[27,28]
Oncogenic signaling-driven immune escape	KRAS/BRAF/MAPK or WNT/β-catenin suppress antigen presentation/chemokines and promote myeloid recruitment	RAS/BRAF mutations; MAPK/WNT activation signatures	Targeted therapy to reprogram TME (e.g., BRAF/MEK/EGFR); rational combinations with ICI[15,26]
Myeloid-dominant suppression (TAM/MDSC)	VEGF/CSF1/IL-8 axis; MDSC expansion; TAM M2 polarization; arginase/ROS-mediated T-cell suppression	High MDSC/TAM signatures; high NLR; VEGF-high tumors	Anti-VEGF/VEGFR TKIs; CSF1R/CXCR1/CXCR2 inhibition; CD47/SIRPα blockade (investigational)[10,16,19]
Checkpoint redundancy and T cell exhaustion	Co-expression of PD-1/PD-L1 with CTLA-4/LAG-3/TIGIT/TIM-3 dysfunctional CD8 pool	Exhaustion signatures; multiple checkpoints on TILs	Dual/next-gen checkpoint blockade; costimulatory agonists; intratumoral delivery[10,14]
Immunometabolic suppression	Hypoxia, lactate; adenosine (CD39/CD73); tryptophan catabolism (IDO/TDO); nutrient competition	High CD73; hypoxia markers; kynurenine/adenosine signatures	A2A/CD73 axis inhibitors; metabolic modulation; normalization of hypoxia/vasculature[35,36]
Microbiome-driven immune modulation	Dysbiosis (e.g., Fusobacterium) shapes myeloid programs and alters T cell function via microbial metabolites	Stool metagenomics; Fusobacterium abundance; bile acid/SCFA profiles	Diet/probiotic/FMT strategies; antibiotic stewardship; microbiome-informed stratification[33,34]
Metastatic niche effects (especially liver)	Hepatic tolerogenic myeloid cells; sequestration/deletion of activated T cells systemic suppression	Presence of liver metastases; low intrahepatic CD8 density	Liver-directed RT/SIRT/TACE + ICI; consider liver metastasis status in trial design[45,66,70]

TGF-β: Transforming growth factor-β; MDSC: Myeloid-derived suppressor cell; TAM: Tumor-associated macrophage; MSS: Microsatellite stable; TMB: Tumor mutational burden; APC: Antigen-presenting cell; B2M: Β2-microglobulin; HLA: Human leukocyte antigen; JAK/STAT: Janus kinase/signal transducer and activator of transcription; IFN: Interferon; CAF: Cancer-associated fibroblast; ECM: Extracellular matrix; MAPK: Mitogen-activated protein kinase; VEGF: Vascular endothelial growth factor; IL: Interleukin; CSF: Colony stimulating factor; PD-1: Programmed cell death protein 1; PD-L1: Programmed death-ligand 1; ROS: Reactive oxygen species; CTLA-4: Cytotoxic T-lymphocyte-associated protein-4; LAG-3: Lymphocyte activation gene-3; TIGIT: T cell immunoglobulin and ITIM domain; CD: Cluster of differentiation; TIM-3: T cell immunoglobulin and mucin domain-containing protein 3; MHC: Major histocompatibility complex; CMS: Consensus molecular subtype; NLR: Neutrophil-to-lymphocyte ratio; TIL: Tumor-infiltrating lymphocyte; SCFA: Short-chain fatty acid; RT: Radiotherapy; STING: Stimulator of interferon genes; TLR: Toll like receptor; TME: Tumor microenvironment; MEK: Mitogen-activated protein kinase kinase; EGFR: Epidermal growth factor receptor; ICI: Immune checkpoint inhibitor; VEGFR: Vascular endothelial growth factor receptor; TKI: Tyrosine kinase inhibitor; SIRPα: Signal regulatory protein alpha; FMT: Fecal microbiota transplantation; SIRT: Selective internal radiation therapy; TACE: Transarterial chemoembolization.

Table 2 Selected clinical evidence for immunotherapy combination strategies in microsatellite-stable/proficient mismatch repair metastatic colorectal cancer

Combination strategy/regimen	Clinical setting (population)	Key outcomes	Predictors/caveats	Ref.
Pembrolizumab + mFOLFOX7 or FOLFIRI (KEYNOTE-651)	1 L/2 L advanced CRC (predominantly pMMR/MSS)	Safety acceptable; no clear signal beyond historical chemo in unselected MSS cohorts	Single-arm; chemotherapy confounds ORR; highlights need for biomarker enrichment	Gallois et al[57]; Kim et al[58]
Maintenance FP + bevacizumab ± atezolizumab (MODUL)	Post-induction maintenance in mCRC (unselected; largely MSS)	No significant PFS (HR = 0.92) or OS (HR = 0.94) improvement vs control	Negative phase II; subgroup signals require validation	Wang et al[59]
CAPOX + bevacizumab + pembrolizumab (FFCD1703-POCHI)	1 L pMMR/MSS mCRC with high TIL/Immunoscore	ORR = 73% (17% CR); DCR 100%; 12-month PFS 52%; 24-month OS 80%	Biomarker-enriched (high Immunoscore); confirm in larger/controlled studies	Yamaguchi et al[60]
Regorafenib + nivolumab (REGONIVO, phase Ib)	Refractory MSS mCRC (Japan)	ORR approximately 33%-36% in CRC cohort; median PFS approximately 7.9 months	Early-phase; later Western studies show lower activity; liver metastases may blunt benefit	Fukuoka et al[65]
Regorafenib + nivolumab (phase II)	Refractory MSS mCRC (multicenter United States)	ORR 7% (5/70 PR); median PFS 1.8 months; OS 11.9 months	All responders had no liver metastases	Fakih et al[66]
Fruquintinib + sintilimab	Refractory MSS mCRC (China phase II)	ORR 12.5%; DCR 76.4%; median PFS 4.1 months; OS 15.3 months	Liver mets: PFS 3.2 months vs 7.6 months; NLR/albumin/ECOG predictive	Zhang et al[68]
Regorafenib + ipilimumab + nivolumab	Refractory MSS mCRC (single-center phase I)	ORR 27.6%; median PFS 4.0 months; OS 20 months; non-liver mets ORR 36.4%	Responses largely confined to nonliver metastatic disease; dose-related skin/immune AEs	Xiao et al[69]; Fakih et al[70]
Atezolizumab + cobimetinib (IMblaze370)	Refractory mCRC (predominantly MSS; phase III)	No OS benefit vs regorafenib; ORR approximately 2%	Suggests MEK inhibition alone is insufficient; scheduling/partner choice critical	Eng et al[40]
Radiotherapy + ICI (various early-phase)	MSS mCRC; often liver-dominant disease	Clinical benefit inconsistent; abscopal responses uncommon	Dose/fractionation, target lesions, and systemic priming likely determinants	Yang et al[91]; Lee et al[92]; Nelson et al[93]; Hang et al[94]
Locoregional therapy + ICI (HIPEC/HAIC/TACE/SIRT; early-phase)	Liver or peritoneal metastases	Primarily early-phase/ongoing; rationale is in situ antigen release and myeloid reprogramming	Safety/sequence critical; endpoints include immune correlatives	Nelson et al[93]; Nevo et al[95]; Jiang et al[96]

CAPOX: Capecitabine + oxaliplatin; FP: Fluoropyrimidine; ICI: Immune checkpoint inhibitor; HIPEC: Hyperthermic intraperitoneal chemotherapy; HAIC: Hepatic arterial infusion chemotherapy; TACE: Transarterial chemoembolization; SIRT: Selective internal radiation therapy; mCRC: Metastatic colorectal cancer; MSS: Microsatellite-stable; pMMR: Mismatch repair-proficient; CRC: Colorectal cancer; TIL: Tumor-infiltrating lymphocyte; OS: Overall survival; PFS: Progression-free survival; CR: Complete response; DCR: Disease control rate; ORR: Objective response rate; HR: Hazard ratio; PR: Partial response; NLR: Neutrophil-to-lymphocyte ratio; ECOG: Eastern Cooperative Oncology Group; AE: Adverse event; MEK: Mitogen-activated protein kinase kinase.

Table 3 Candidate biomarkers for patient selection and monitoring in microsatellite-stable/proficient mismatch repair colorectal cancer

Biomarker	Assay/readout	Rationale in MSS CRC	Clinical application and limitations	Ref.
MSIH/dMMR	IHC (MLH1/MSH2/MSH6/PMS2), PCR, NGS	High neoantigen load and inflamed TME	Established selection for PD-1 (± CTLA-4); rare in metastatic CRC	Gandini et al[130]; Ambrosini et al[131]
TMB-high; POLE/POLD1 ultra-mutation	NGS panel; TMB cutoffs	May confer immunogenicity despite MSS	Identifies small ICI-sensitive subset; platform/cutoff heterogeneity	Xue et al[132]; Domingo et al[135]
PD-L1 expression	IHC (TPS/CPS; immune cell staining)	Surrogate of immune activation, but weak predictor in CRC	Limited standalone utility; may contribute in combination contexts	Nakamura et al[137]
Immunoscore/TIL density (CD3/CD8)	Standardized IHC quantification (center + invasive margin)	Captures pre-existing antitumor immunity; enriches for ICI-responsive biology	Potential for patient selection (e.g., POCHI); requires harmonization and cutoffs	Yamaguchi et al[60]; Esmail et al[134]
Inflamed vs excluded gene signatures (IFN-γ; TGF-β/stromal)	RNA-seq/nano string signatures	Defines immune-hot vs immune-excluded states and matches mechanisms	Trial stratification; resource-intensive; signatures not yet standardized	Mortezaee and Majidpoor[27]; Li et al[28]; Esmail et al[134]
Liver metastasis status	Imaging; metastatic pattern	Liver promotes systemic immune tolerance and attenuates ICI benefit	Negative predictor in multiple ICI + TKI datasets; guides patient selection/Locoregional consolidation	Fakih et al[66]; Fakih et al[70]; Taïeb et al[138]
ctDNA kinetics	Plasma NGS (VAF dynamics; MRD)	Early molecular response/resistance signal	Monitoring and adaptive strategies; assay and threshold variability	Hou et al[140]
Host inflammatory/nutrition indices	NLR, albumin, ECOG	Correlates with myeloid skewing and immune fitness	Adjunct prognostic/predictive markers (not standalone)	Zhang et al[68]
Gut microbiome features	Metagenomics; targeted qPCR (e.g., Fusobacterium)	Microbiota shapes systemic immunity and therapy response/toxicity	Exploratory; actionable interventions (diet/FMT) under evaluation	Luo et al[33]; Luu et al[34]; Ramachandran et al[139]

MSI-H: Microsatellite instability-high; dMMR: Deficient mismatch repair; TMB: Tumor mutational burden; PD-L1: Programmed death-ligand 1; TIL: Tumor-infiltrating lymphocyte; CD: Cluster of differentiation; IFN: Interferon; TGF-β: Transforming growth factor-β; ctDNA: Circulating tumor DNA; IHC: Immunohistochemistry; NGS: Next-generation sequencing; PCR: Polymerase chain reaction; qPCR: Quantitative polymerase chain reaction; CPS: Combined positive score; TPS: Tumor proportion score; RNA-seq: RNA-sequencing; MRD: Minimal residual disease; VAF: Variant allele frequency; NLR: Neutrophil-to-lymphocyte ratio; ECOG: Eastern Cooperative Oncology Group; TME: Tumor microenvironment; MSS: Microsatellite-stable; CRC: Colorectal cancer; ICI: Immune checkpoint inhibitor; PD-1: Programmed cell death protein 1; CTLA-4: Cytotoxic T-lymphocyte-associated protein-4; TKI: Tyrosine kinase inhibitor; FMT: Fecal microbiota transplantation.