Abstract Background: High-dose vitamin C (VitC) has shown superior anticancer activity in immunocompetent compared with immunodeficient mice, suggesting an immune-dependent mechanism of action. Because high-dose VitC generates substantial reactive oxygen species (ROS), we hypothesized that ROS might induce immunogenic cell death (ICD)—a regulated form of cell death characterized by the release of damage-associated molecular patterns (DAMPs) that enhance antigen presentation and promote antitumor immunity. Methods: Spatial transcriptomic and immunofluorescence analyses were performed on breast and colon tumors from mice treated with high-dose VitC to quantify oxidative stress, DAMP mobilization, and immune cell infiltration. To dissect the functional role of ROS and ICD, mice were treated with high-dose VitC in combination with the antioxidant N-acetylcysteine or an antibody blocking calreticulin (an early DAMP essential for ICD induction). Results: VitC treatment caused marked oxidative stress in tumors from both immunocompetent and immunocompromised mice; however, tumor growth inhibition occurred exclusively in immunocompetent animals. High-dose VitC triggered ROS-dependent exposure and release of the DAMPs calreticulin and HMGB1, respectively. This was accompanied by substantial remodeling of the tumor microenvironment, including increased infiltration of cytotoxic CD8+ T cells and natural killer cells, and a reduction in immunosuppressive regulatory T cells. Blocking calreticulin effectively disrupted the ICD cascade, prevented immune microenvironment remodeling, and completely abrogated the antitumor efficacy of VitC. Conclusions: High-dose VitC promotes ICD through ROS-dependent mobilization of calreticulin and HMGB1, and calreticulin is required for its immune-mediated antitumor activity. These results provide a mechanistic rationale for ongoing translational analyses within the ALFEO clinical trial (ECTR2022-502101-15-00), which is evaluating high-dose VitC in combination with nivolumab and ipilimumab in mismatch-repair-proficient colorectal cancer. Funded by the Italian Ministry of Health Next Generation EU - PNRR M6C2 - PNRR-MAD-2022-12376593. Citation Format: Alessandro Cavaliere, Federica Maione, Marco Macagno, Vito Amodio, Rosaria Chilà, Marcello Turi, Marc Escobosa, Giovanni Germano, Simona Lamba, Chiara Baretta, Anita Brignacca, Valeria Pessei, Elena Perez, Daniela Grases, Alice Bartolini, Fabio Penna, Eduard Porta, Manel Esteller, Dieter Saur, Roland Rad, Annamaria Gullà, Alberto Bardelli, Teresa Troiani, Salvatore Siena, Andrea Sartore-Bianchi, Federica Di Nicolantonio. High-dose vitamin C induces ROS-dependent calreticulin exposure to drive immunogenic cell death and cancer immune surveillance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4298.

We previously reported that high-dose Vitamin C (VitC) can synergize with immune checkpoint blockade in immunocompetent mice bearing colorectal, pancreatic and breast tumors. Here, we aim to unveil the molecular mechanisms underlying the above preclinical observations. We hypothesized that VitC could trigger immunogenic cell death (ICD), thereby stimulating an immune-mediated anticancer response. Induction of ICD was assessed by the expression or emission of specific markers, including calreticulin, as well as by ex-vivo phagocytosis assays by co-culturing dendritic cells with breast or colon cancer cells treated with VitC. A calreticulin blocking antibody was administered in vivo in combination with VitC to functionally characterize the role of ICD induction in the anticancer effects of vitC. Ex-vivo spatial transcriptomic and immunofluorescence analyses were performed to characterize the impact of the treatment on tumor and immune cell populations. Pharmacological doses of VitC were able to induce oxidative stress as indicated by spatial transcriptomic data and confirmed by ex vivo 8-hydroxyguanine staining in tumor sections. VitC treatment induced ICD markers and among these calreticulin and HMGB1. Although the induction of these danger markers was seen in tumors grown both in immunocompromised and immunocompetent VitC treated mice, anticancer activity was evident only in the presence of an intact immune system. In vivo, a calreticulin blocking antibody effectively prevented its translocation and inhibited HMGB1 release in murine tumors. Notably, preventing calreticulin exposure abrogated the immune mediated anticancer effect of VitC. VitC enhanced the infiltration of natural killers, activated CD8+ T lymphocytes, while it decreased the number of Tregs. Calreticulin blockade impaired the modulation of the immune tumor microenvironment induced by VitC. VitC treatment can mobilize calreticulin and HMGB1, which are among the established markers of immunogenic cell death. These results, alongside the observation that VitC exerts its maximal antitumor activity only in immunocompetent mice and its anticancer activity is abolished by anti-calreticulin blocking antibody demonstrated that VitC is an ICD inducer when given at pharmacological doses. These findings lend the preclinical rationale and inform the translational analyses planned within the ALFEO clinical trial (Clinical trial information: ECTR2022-502101-15-00), that is investigating pharmacological doses of vitC in combination with nivolumab and ipilimumab in mismatch repair proficient colon cancer patients. Funded by- Next Generation EU - PNRR M6C2 - PNRR-MAD-2022-12376593. Alessandro Cavaliere, Marco Macagno, Federica Maione, Vito Amodio, Marc Escobosa Olmo, Rosaria Chilà, Simona Lamba, Pietro Paolo Vitiello, Valeria Pessei, Daniela Grases, Elena Perez, Alice Bartolini, Noemi Congiusta, Giovanni Germano, Andrea Sartore-Bianchi, Teresa Troiani, Manel Esteller, Eduard Porta, Alberto Bardelli, Federica Di Nicolantonio. Immunogenic cell death mediates the anticancer efficacy of high dose vitamin C [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6169.

Efficacy of targeted therapies in metastatic colorectal cancer (mCRC) is hindered by onset of resistance, therefore novel strategies are needed. A subpopulation of drug-tolerant persister cells (DTPs) survives to targeted therapies and initiates adaptive mutability response. DTPs experience an increased DNA damage and a shift to error-prone DNA damage repair, thus promoting mutability and resistance. The purpose of this project is to explore key players modulating adaptive mutability to prevent DTPs’ evolution to resistance, thus preventing tumor recurrence. CAS9-expressing CRC cells were infected with a custom CRISPR library targeting over 500 DNA damage response (DDR) genes, divided in 12 subpopulations, and grown either for 4 weeks in normal cell culture conditions (untreated) or treated with specific oncogenic inhibitors until the development of resistance in a Time-To-Progression (TTP) assay. CAS9-expressing clones infected with empty library were grown in parallel, in absence or presence of targeted therapies, as control. Next Generation Sequencing-based analysis of sgRNA (single guides RNA) was then performed on resistant subpopulations grown in the presence of targeted therapies. We initially isolated CAS9-expressing clones from a microsatellite stable (MSS) CRC cell line and selected clones with growth rate and drug-sensitivity comparable to that of parental population. The selected CAS9-expressing clone was then infected with the CRISPR DDR library or empty library and, after the selection with puromycin, the selected populations were divided in 12 subpopulations, to exclude the possibility that pre-existing resistant cells might outgrow and interfere with the effect of DDR genes knock-outs (KO) on the development of drug-resistance. The subpopulations were then grown in absence or presence of oncogenic inhibitors for several weeks until the onset of resistance. Notably, while the 12 subpopulations infected with the empty library simultaneously developed resistance, the development of resistance in the DDR library-infected cells was spread over multiple weeks, thus highlighting that KO of DDR genes might either accelerate or delay the onset of resistance. sgRNA of resistant population were then analyzed by next-generation sequencing. A negative selection analysis will be applied to identify sgRNA that are lost in the resistant populations, thus targeting DDR genes whose depletion is potentially synthetical lethal with targeted therapies in mCRC. A DDR CRISPR screening identified novel vulnerabilities of CRC DTPs able to improve efficacy of targeted therapies and prevent tumor relapse in mCRC. Martina Miotto, Simona Lamba, Gaia Grasso, Giorgio Corti, Julie Bonetto, Alberto Sogari, Alberto Bardelli, Mariangela Russo. CRISPR-CAS9 library screening identifies novel vulnerabilities in CRC drug-tolerant persister cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4190.

Hypermutation induced by mismatch repair (MMR) inactivation leads to immune surveillance in colorectal cancer (CRC) and in several other malignancies. We investigated the impact of a rationally designed chemotherapy combination on the generation of hypermutation and immunogenicity in otherwise immune-refractory CRC and breast cancer mouse models. Combinatorial treatment with cisplatin (CDDP) and temozolomide (TMZ) induces an adaptive downregulation of MMR, resulting in chemotherapy-dependent hypermutability and increase in predicted neoantigens. This combination specifically alters the immune fitness of the tumors, ultimately leading to CD8+ T cell-mediated immune surveillance, immunoediting of chemotherapy-induced neoantigens, and durable immunological memory. Treatment with CDDP and TMZ also remodels the innate immune microenvironment and induces long-lasting responses and complete rejections when combined with anti-PD-1 therapy in mice. The same effects are not observed using the clinically approved combination of 5-fluorouracil, oxaliplatin, and irinotecan (FOLFOXIRI). Treatment-induced hypermutation can enhance anti-tumor immune responses, offering additional avenues for cancer treatment.

Background Immunotherapy based on checkpoint inhibitors is highly effective in mismatch repair deficient (MMRd) colorectal cancer (CRC). These tumors carry a high number of mutations, which are predicted to translate into a wide array of neoepitopes; however, a systematic classification of the neoantigen repertoire in MMRd CRC is lacking. Mass spectrometry peptidomics has demonstrated the existence of MHC class I associated peptides (MAPs) originating from non-coding DNA regions. Based on these premises we investigated DNA genomic regions responsible for generating MMRd-induced peptides. Methods We exploited mouse CRC models in which the MMR gene Mlh1 was genetically inactivated. Isogenic cell lines CT26 Mlh1 +/+ and Mlh1 -/- were inoculated in immunocompromised and immunocompetent mice. Whole genome and RNA sequencing data were generated from samples obtained before and after injection in murine hosts. First, peptide databases were built from transcriptomes of isogenic cell lines. We then compiled a database of peptides lost after tumor cells injection in immunocompetent mice, likely due to immune editing. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and matched next-generation sequencing databases were employed to identify the DNA regions from which the immune-targeted MAPs originated. Finally, we adopted in vitro T cell assays to verify whether MAP-specific T cells were part of the in vivo immune response against Mlh1 -/- cells. Results Whole genome sequencing analyses revealed an unbalanced distribution of immune edited alterations across the genome in Mlh1 -/- cells grown in immunocompetent mice. Specifically, untranslated (UTR) and coding regions exhibited the largest fraction of mutations leading to highly immunogenic peptides. Moreover, the integrated computational and LC-MS/MS analyses revealed that MAPs originate mainly from atypical translational events in both Mlh1 +/+ and Mlh1 -/- tumor cells. In addition, mutated MAPs—derived from UTRs and out-of-frame translation of coding regions—were highly enriched in Mlh1 -/- cells. The MAPs trigger T-cell activation in mice primed with Mlh1 -/- cells. Conclusions Our results suggest that—in comparison to MMR proficient CRC—MMRd tumors generate a significantly higher number of non-canonical mutated peptides able to elicit T cell responses. These results reveal the importance of evaluating the diversity of neoepitope repertoire in MMRd tumors.

Serine/threonine‐protein kinase B‐raf ( BRAF ) mutations are found in 8–15% of colorectal cancer patients and identify a subset of tumors with poor outcome in the metastatic setting. We have previously reported that BRAF‐ mutant human cells display a high rate of protein production, causing proteotoxic stress, and are selectively sensitive to the proteasome inhibitors bortezomib and carfilzomib. In this work, we tested whether carfilzomib could restrain the growth of BRAF‐ mutant colorectal tumors not only by targeting cancer cells directly, but also by promoting an immune‐mediated antitumor response. In human and mouse colorectal cancer cells, carfilzomib triggered robust endoplasmic reticulum stress and autophagy, followed by the emission of immunogenic‐damage‐associated molecules. Intravenous administration of carfilzomib delayed the growth of BRAF‐ mutant murine tumors and mobilized the danger‐signal proteins calreticulin and high mobility group box 1 (HMGB1). Analyses of drug‐treated samples revealed increased intratumor recruitment of activated cytotoxic T cells and natural killers, concomitant with the downregulation of forkhead box protein P3 (Foxp3) + T‐cell surface glycoprotein CD4 (CD4) + T cells, indicating that carfilzomib promotes reshaping of the immune microenvironment of BRAF‐ mutant murine colorectal tumors. These results will inform the design of clinical trials in BRAF‐ mutant colorectal cancer patients.

Abstract Background Liquid biopsy based on cell-free DNA (cfDNA) analysis holds significant promise as a minimally invasive approach for the diagnosis, genotyping, and monitoring of solid malignancies. Human tumors release cfDNA in the bloodstream through a combination of events, including cell death, active and passive release. However, the precise mechanisms leading to cfDNA shedding remain to be characterized. Addressing this question in patients is confounded by several factors, such as tumor burden extent, anatomical and vasculature barriers, and release of nucleic acids from normal cells. In this work, we exploited cancer models to dissect basic mechanisms of DNA release. Methods We measured cell loss ratio, doubling time, and cfDNA release in the supernatant of a colorectal cancer (CRC) cell line collection (N = 76) representative of the molecular subtypes previously identified in cancer patients. Association analyses between quantitative parameters of cfDNA release, cell proliferation, and molecular features were evaluated. Functional experiments were performed to test the impact of modulating DNA methylation on cfDNA release. Results Higher levels of supernatant cfDNA were significantly associated with slower cell cycling and increased cell death. In addition, a higher cfDNA shedding was found in non-CpG Island Methylator Phenotype (CIMP) models. These results indicate a positive correlation between lower methylation and increased cfDNA levels. To explore this further, we exploited methylation microarrays to identify a subset of probes significantly associated with cfDNA shedding and derive a methylation signature capable of discriminating high from low cfDNA releasers. We applied this signature to an independent set of 176 CRC cell lines and patient derived organoids to select 14 models predicted to be low or high releasers. The methylation profile successfully predicted the amount of cfDNA released in the supernatant. At the functional level, genetic ablation of DNA methyl-transferases increased chromatin accessibility and DNA fragmentation, leading to increased cfDNA release in isogenic CRC cell lines. Furthermore, in vitro treatment of five low releaser CRC cells with a demethylating agent was able to induce a significant increase in cfDNA shedding. Conclusions Methylation status of cancer cell lines contributes to the variability of cfDNA shedding in vitro. Changes in methylation pattern are associated with cfDNA release levels and might be exploited to increase sensitivity of liquid biopsy assays.

Abstract Introduction. The bacterial genotoxin colibactin is enriched in colorectal cancer (CRC) and promotes the accumulation of mutations that drive tumorigenesis. However, systematic assessment of its impact on DNA damage response is lacking and the effect of colibactin exposure on response to other genotoxic agents (such as chemotherapy) is missing. Materials and methods: We implemented an in vitro bacteria-coculture system to assess the effect of colibactin on a representative subset of 40 molecularly and pharmacologically annotated CRC cell lines and in a panel of isogenic DDR KO cell lines we generated. We further validated our results in patient-derived organoids. Finally, we recapitulated prolonged exposure to colibactin occurring during tumorigenesis by chronically infecting sensitive cells until the emergence of a tolerant phenotype. Results: We found that different cell lines display specific sensitivity to colibactin’s genotoxic stress: while colibactin-tolerant cells are capable of quickly and efficiently repairing colibactin-induced DNA damage, sensitive cells lack this ability. Moreover, we found that homologous recombination (HR) proficiency discriminates colibactin-tolerant cells, which display higher levels of RAD51 foci (as marker of activation of HR) compared to sensitive cells upon infection with colibactin. Screening of isogenic DDR KO cell lines revealed that genetic inactivation of the intertwined pathways of HR (through KO of ATM) and replication stress (RS) response (through KO of ATRIP) significantly sensitized cells to colibactin. In addition, we found that restoration of HR activity was sufficient to induce a colibactin-tolerant phenotype in initially sensitive cell lines. Notably, thanks to a previous effort of pharmacological characterization of CRC cell lines in our lab, we found a significant correlation between sensitivity to colibactin and irinotecan active metabolite SN38, but not oxaliplatin. We validated the same correlation in patient-derived organoids annotated for response to SN38. While colibactin, SN38 and oxaliplatin all induced RS in treated cells, we found that colibactin and SN38 showed a similar DNA damage response which involved activation of ATM. Finally, chronic re-infection of sensitive, HR-deficient CRC cells with colibactin selected a tolerant phenotype characterized by restoration of HR activity. Of translational relevance, colibactin-tolerant derivative cells acquired cross-resistance to SN38 and PARP inhibitor olaparib but not to oxaliplatin. Conclusion: Our results shed novel insight into colibactin’s genotoxic mechanism and support a model in which colibactin both promotes tumorigenesis and acts as an evolutionary bottleneck which selects HR proficient CRC cells. Furthermore, our study provides preclinical evidence on colibactin’s role in promoting chemoresistance in colorectal cancer. Citation Format: Alberto Sogari, Emanuele Rovera, Nicole Megan Reilly, Simona Lamba, Erika Durinikova, Annalisa Lorenzato, Marco Avolio, Eleonora Piumatti, Mariangela Russo, Sabrina Arena, Livio Trusolino, Manuela Donalisio, Federica Di Nicolantonio, David Lembo, Alberto Bardelli. Tolerance to colibactin correlates with response to chemotherapeutic agents in colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr B005.

The bacterial genotoxin colibactin promotes colorectal cancer (CRC) tumorigenesis, but systematic assessment of its impact on DNA repair is lacking, and its effect on response to DNA-damaging chemotherapeutics is unknown. We find that CRC cell lines display differential response to colibactin on the basis of homologous recombination (HR) proficiency. Sensitivity to colibactin is induced by inhibition of ATM, which regulates DNA double-strand break repair, and blunted by HR reconstitution. Conversely, CRC cells chronically infected with colibactin develop a tolerant phenotype characterized by restored HR activity. Notably, sensitivity to colibactin correlates with response to irinotecan active metabolite SN38, in both cell lines and patient-derived organoids. Moreover, CRC cells that acquire colibactin tolerance develop cross-resistance to SN38, and a trend toward poorer response to irinotecan is observed in a retrospective cohort of CRCs harboring colibactin genomic island. Our results shed insight into colibactin activity and provide translational evidence on its chemoresistance-promoting role in CRC.

The introduction of targeted therapies represented one of the most significant advances in the treatment of BRAFV600E melanoma. However, the onset of acquired resistance remains a challenge. Previously, we showed in mouse xenografts that vascular endothelial growth factor (VEGFA) removal enhanced the antitumor effect of BRAF inhibition through the recruitment of M1 macrophages. In this work, we explored the strategy of VEGFA/BRAF inhibition in immunocompetent melanoma murine models. In BRAF mutant D4M melanoma tumors, VEGFA/BRAF targeting reshaped the tumor microenvironment, largely by stimulating infiltration of M1 macrophages and CD8 + T cells, and sensitized tumors to immune checkpoint blockade (ICB). Furthermore, we reported that the association of VEGFA/BRAF targeting with anti‐PD‐1 antibody (triple therapy) resulted in a durable response and enabled complete tumor eradication in 50% of the mice, establishing immunological memory. Neutralization and CRISPR‐Cas‐mediated editing of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) abrogated antitumor response prompted by triple therapy and identified GM‐CSF as the cytokine instrumental in M1‐macrophage recruitment. Our data suggest that VEGFA/BRAF targeting in melanoma induces the activation of innate and adaptive immunity and prepares tumors for ICB. Our study contributes to understanding the tumor biology of BRAFV600E melanoma and suggests VEGFA as therapeutic target.

Stroma NCL inhibition increases pericyte coverage of tumor blood vessels.

The supplementary material includes Supplementary Methods, two Supplementary Tables, and six Supplementary Figures Table S1. Complete set of BCAM-mimic peptides. Table S2. Characterization of human CRC cell lines in animal models. Figure S1. Quality control of the phage display experiments: sequencing readout. Figure S2. BCAM protein expression in the complete panel of human hepatic metastases. Figure S3. BCAM protein expression in human primary CRCs. Figure S4. BCAM protein and mRNA expression in all cell lines. Figure S5. Photographic documentation of the experimental hepatic metastasis models. Figure S6. HT-55, DLD-1, LIM1215 and SW-48 cells cannot be reduced to adhesion-functional monocellular suspensions.

mPDAC orthopic model characterization.

N6L and angiogenic molecules regulation.

N6L inhibits PDAC proliferation and migration

PDF file - 161K, Ctrl and MLK4 knock down DLD were seeded and allowed to grow for the indicated times in flasks with ultra-low attachment surface to prevent cell adhesion. Cells were harvested and protein lysates were subjected to western blotting with the indicated antibodies.

AbstractMost patients with KRASG12C–mutant non–small cell lung cancer (NSCLC) experience clinical benefit from selective KRASG12C inhibition, whereas patients with colorectal cancer bearing the same mutation rarely respond. To investigate the cause of the limited efficacy of KRASG12C inhibitors in colorectal cancer, we examined the effects of AMG510 in KRASG12C colorectal cancer cell lines. Unlike NSCLC cell lines, KRASG12C colorectal cancer models have high basal receptor tyrosine kinase (RTK) activation and are responsive to growth factor stimulation. In colorectal cancer lines, KRASG12C inhibition induces higher phospho-ERK rebound than in NSCLC cells. Although upstream activation of several RTKs interferes with KRASG12C blockade, we identify EGFR signaling as the dominant mechanism of colorectal cancer resistance to KRASG12C inhibitors. The combinatorial targeting of EGFR and KRASG12C is highly effective in colorectal cancer cells and patient-derived organoids and xenografts, suggesting a novel therapeutic strategy to treat patients with KRASG12C colorectal cancer.Significance:The efficacy of KRASG12C inhibitors in NSCLC and colorectal cancer is lineage-specific. RTK dependency and signaling rebound kinetics are responsible for sensitivity or resistance to KRASG12C inhibition in colorectal cancer. EGFR and KRASG12C should be concomitantly inhibited to overcome resistance to KRASG12C blockade in colorectal tumors.See related commentary by Koleilat and Kwong, p. 1094.This article is highlighted in the In This Issue feature, p. 1079

NCL blocking antibody and Ncl deletion affects PDAC cell proliferation.

Supplementary Table from Targeting the DNA Damage Response Pathways and Replication Stress in Colorectal Cancer

Supplementary Table 1 from Novel Somatic and Germline Mutations in Cancer Candidate Genes in Glioblastoma, Melanoma, and Pancreatic Carcinoma