From Gate-Jamming Score to Gate-Opening Sequence for MSS Colorectal Cancer
Anthony J. Vasquez Sr. | Delaware Valley University | February 2026 | v4.0
Preprint: Context-Specific Innate Immune Evasion via VDAC1 Gate-Jamming in Microsatellite-Stable Colorectal Cancer (Research Square, 2026) -- Transcriptomic validation across TCGA pan-cancer (n=10,071), COADREAD MSS/TP53-wt clean room (n=209), and IMvigor210 (n=348). Analysis code and data: templetwo/vdac-pharmacology-atlas
Companion work: CBD's Paradox at the Mitochondrial Gate (OSF Preprints, 2026) -- Multi-LLM convergence study of VDAC1 pharmacology (78 references)
Microsatellite-stable (MSS) colorectal cancer is immune-invisible. Checkpoint inhibitors that work in MSI-H tumors fail completely here. This repository contains the Strategic Architecture Document (SAD v4) describing a reformulated therapeutic hypothesis targeting the VDAC1 channel on the outer mitochondrial membrane.
The original three-phase hypothesis (TSPO inhibition → immune activation → CBD apoptosis) was killed by its own literature review. Three fatal contradictions were identified. What survived is mechanistically cleaner and clinically actionable:
Lovastatin (cholesterol lock)
→ VDAC1 oligomerization → mtDNA release → cGAS-STING activation
→ Botensilimab amplifies innate → adaptive immune response
The apoptotic threshold of a cancer cell is governed by three physical locks on VDAC1:
Cancer rewrites all three terms simultaneously. Each lock requires a specific key. In MSS CRC, the rate-limiting lock is the cholesterol-to-cardiolipin ratio in the OMM lipid annulus.
The preprint operationalizes the biophysical GJS as a transcriptomic score for TCGA analysis:
In the MSS/TP53-wildtype COADREAD clean room (n=209), high tGJS was inversely correlated with HAVCR2/TIM-3 (
| Finding | Implication |
|---|---|
| Lovastatin induces mtDNA release + cGAS-STING in HCT116 (Huang et al., 2024) | Collapses original Phase 1+2 into one mechanistic step |
| Statins deplete mitochondrial membrane cholesterol (Shen et al., 2024) | Lipid-annulus mechanism is defensible |
| CBD suppresses T-cells at VDAC1-active doses | CBD formally quarantined from immune window |
| TSPO physically tethers VDAC1 (Gatliff et al., 2014) | Reframed as explorable fourth lock, not load-bearing |
| Statin + ICI: 20% mortality reduction (Liao et al., 2025; n=46,154) | Retrospective support for combination |
docs/
SAD_v4_VDAC1_Gate_Opening_Stack.pdf # Current version (complete bench protocol)
archive/
SAD_v3_VDAC1_Gate_Opening_Stack.md # v3: 6 experiments, reagent specs, timeline
SAD_v3_VDAC1_Gate_Opening_Stack.docx
SAD_v2_VDAC1_Gate_Opening_Stack.md # v2: added TSPO cautions, expanded tables
SAD_v2_VDAC1_Gate_Opening_Stack.docx
SAD_v1_VDAC1_Gate_Opening_Stack.md # v1: original 4-experiment architecture
SAD_v1_VDAC1_Gate_Opening_Stack.docx
simulations/ # GJS simulation & Chou-Talalay analysis
experiments/ # Experiment protocols & results (future)
references/ # Key citations & supplementary material
figures/ # Diagrams & visualizations
correspondence/ # Faculty & collaboration letters
wiki/ # Documentation pages (11 topics)
Six experiments with explicit kill conditions are specified in the SAD. The dependency structure:
Exp 2a (OMM cholesterol) ──→ Exp 2b (VDAC1 oligomerization) ←── LINCHPIN
↓
Exp 3 (mtDNA → cGAS-STING)
↓
Exp 4 (CT26 mouse in vivo)
Exp 1 (TSPO tether) ──→ Exp 5 (lovastatin + PK11195 synergy)
Exp 6 (CBD disposition) ──→ CBD quarantine decision
Experiment 2a/2b is the linchpin. If lovastatin does not deplete OMM cholesterol and trigger VDAC1 oligomerization in HCT116 cells, the entire framework collapses. Four weeks. $3,010. Everything depends on this result.
| Phase | Window | Goal | Deliverable |
|---|---|---|---|
| A | Feb–Mar 2026 | OSF registration + public GitHub release | DOI: 10.17605/OSF.IO/4KNQR |
| B | Apr–Jun 2026 | AML venetoclax temporal-decoupling (proves physics) | Gate-opener drug class validation |
| C | Jul–Sep 2026 | HCT116 lovastatin Exp 2a+2b (LINCHPIN) | Mechanistic confirmation |
| D | Oct–Dec 2026 | TSPO tether + synergy + CT26 mouse model | In vivo efficacy + journal submission |
| E | Parallel w/ C | CBD disposition (Exp 3 co-culture) | Quarantine decision |
9-month sprint. Standard reagents. Single-PI compatible.
SAD v4 includes a complete bench-ready protocol for the linchpin experiment:
- Cell line: HCT116 (MSS CRC, TP53-wt, ATCC CCL-247)
- Lovastatin activation: Prodrug conversion via NaOH hydrolysis, neutralization, filter sterilization
- Readouts: Amplex Red cholesterol quantification, filipin staining, DSS cross-linking + Western blot (VDAC1 oligomerization), qPCR cytosolic mtDNA, IFN-β ELISA
- Controls: VDAC1-KO, MβCD positive control, vehicle
- Budget: $3,010 | Timeline: 4 weeks
- Statistical plan: One-way ANOVA with Tukey post-hoc, α = 0.05, n ≥ 3
- Literature review and falsification of original hypothesis
- Reformulated therapeutic stack architecture (SAD v4)
- Falsification triggers defined for each component
- Six critical experiments designed with kill conditions
- Complete experiment table with reagents and cell lines
- Validated support mechanisms with evidence grades
- Complete repository with indexes, wiki, 90-reference bibliography
- OSF preregistration (DOI: 10.17605/OSF.IO/4KNQR)
- Phase A: Public release (Feb 23, 2026)
- Preprint posted to Research Square (Feb 26, 2026; DOI: 10.21203/rs.3.rs-8935902/v1)
- Phase B: AML venetoclax temporal-decoupling experiment
- Phase C: HCT116 lovastatin gate-opening experiments (2a + 2b)
- Phase D: TSPO exploration + CT26 mouse model
- Phase E: CBD disposition via co-culture experiment
Cite the preprint:
@article{vasquez2026vdac1_preprint,
title={Context-Specific Innate Immune Evasion via VDAC1 Gate-Jamming
in Microsatellite-Stable Colorectal Cancer},
author={Vasquez, Anthony J.},
year={2026},
doi={10.21203/rs.3.rs-8935902/v1},
url={https://doi.org/10.21203/rs.3.rs-8935902/v1},
journal={Research Square (Preprint)},
institution={Delaware Valley University}
}Cite this repository (experimental architecture):
@article{vasquez2026vdac1_sad,
title={VDAC1 Gate-Opening Therapeutic Stack for MSS Colorectal Cancer:
From Gate-Jamming Score to Gate-Opening Sequence},
author={Vasquez, Anthony J., Sr.},
year={2026},
doi={10.17605/OSF.IO/4KNQR},
url={https://osf.io/yn3dw},
note={Strategic Architecture Document v4.0},
institution={Delaware Valley University}
}This work is licensed under CC BY-NC 4.0. You may share and adapt this material for non-commercial purposes with attribution.
The literature burned the old phase sequence, but it handed us a weaponized, clinically actionable stack.
Silence has a measurable geometry. Now we know how to break it.