Introduction

Reovirus, a naturally occurring oncolytic virus, triggers the lysis of tumor cells while also releasing tumor antigens or proapoptotic cytokines into the tumor microenvironment to enhance anticancer immunity. However, reovirus has developed a strategy to evade antiviral immunity by inhibiting interferon production, which negatively affects the induction of antitumor immune responses. The mammalian adaptor protein Stimulator of Interferon Genes (STING) has been identified as a crucial regulator that orchestrates immune responses by sensing cytosolic DNA derived from pathogens or tumors, resulting in the production of type I interferon. Thus, we hypothesize that activation of the STING axis can counteract the suppression of type I IFN responses by reovirus, and combination treatment with reovirus and a STING agonist can promote anticancer immunity by improving the tumor microenvironment. In this study, we investigated the effects of combining reovirus with a STING agonist on antitumor immunity.

Aims & Methods

To investigate the role of immune responses in primary tumor growth inhibition, we performed a dual flank study using an immunocompetent syngeneic model. In the dual flank syngeneic mouse tumor model, CT26 cells were implanted on both sides of a mouse, and then one tumor was injected with reovirus, ADU-S100, or reovirus and ADU-S100, while the other tumor remained untreated. To further verify the immunotherapeutic effect of the T cell infiltration in tumor tissues of mice receiving the combination treatment, we also performed the tumor-infiltrating lymphocytes profiling in the tumor tissues of mice with the treatment using fluorescence-activated cell sorting (FACS).

Results

The combination treatment of reovirus and ADU-S100 induced significant regression in the treated tumor and the untreated distant tumor, indicating that the combination treatment activated a systemic immune response in the dual-flank tumor model. However, intratumoral (IT) injection of reovirus or ADU-S100 alone did not significantly delay tumor growth in the untreated distant tumor. The combination treatment also significantly prolonged the survival of mice bearing dual tumors. The tumor-infiltrating lymphocyte profiling showed a significantly lower CD4:CD8 T cell ratio in the treated and untreated tumors from CT26 tumor-bearing mice treated with the combination IT injection of reovirus and ADU-S100, compared to the untreated controls. However, there were no significant differences in the CD4:CD8 ratio between the control and other treatment groups. We also detected a significant reduction in the ratio of regulatory T cells in tumors treated with reovirus alone or the combination of reovirus and ADU-S100 compared to the control.

Conclusion

We demonstrated the ability of oncolytic reovirus to induce tumor regression in a CT26 syngeneic tumor model and showed that the combination treatment of reovirus and a STING agonist enhanced antitumor immunity. This enhanced antitumor response was partially mediated by IFN-induced T cell activation. Although further investigation is required to clarify the mechanism underlying the synergy between STING agonist and reovirus in promoting immune responses and anti-tumor activity, our combination treatment strategy may have potential as an immunotherapy against colorectal cancer.

Introduction

As the third leading malignancy worldwide, colorectal cancer (CRC) demonstrates strong associations with gut microbiota dysbiosis. Here we investigate, as a key commensal bacterium, how Bifidobacterium breve (B. breve) secreted exopolysaccharides (EPS) modulate anti-tumor immunity through T cell reprogramming.

Aims & Methods

Clinical B. breve abundance was quantified through analysis of patient datasets. Preclinical validation employed three models: 1. CT26 xenografts in NSG vs. BALB/c mice; 2. AOM/DSS-induced colitis-associated carcinoma; 3. EPS bioactivity identification via size-exclusion chromatography and thermal denaturation assays. Immunological effects were analysed through flow cytometry, focusing on stemness-associated markers and exhaustion markers. Systemic immune tracking was mapped across mesenteric lymph nodes, spleen, and tumors.

Results

Metagenomic analysis identified a marked enrichment of Bifidobacterium breve in colorectal cancer (CRC) patients. Subsequent investigation in AOM/DSS-induced CRC murine models demonstrated that oral administration of B. breve substantially attenuated tumor progression. Flow cytometric profiling of tumor infiltrates revealed enhanced T cell stemness and mitigated T cell exhaustion upon bacterial intervention, indicative of functional T cell rejuvenation and altered immunosuppressive microenvironment dynamics. Through systematic fractionation of bacterial supernatants in T cell co-culture systems, we determined that the immunomodulatory activity resides specifically in heat-stable, high-molecular-weight (>3 kDa) components, characteristics aligning with extracellular polysaccharides (EPS). Validation experiments in both NSG immunodeficient and wild-type immunocompetent xenograft models confirmed EPS-mediated antitumor effects require intact immune competence. While oral EPS administration cannot directly transgress the intestinal barrier, comprehensive immune monitoring across mesenteric lymph nodes, splenic tissue, and distal tumor sites revealed coinstantaneous enhancement of T cell stemness profiles, suggesting lymphoid-mediated systemic immunosurveillance potentiation. Notably, EPS supplementation synergized with anti-PD-L1 checkpoint blockade, proposing a microbiota-derived strategy to potentiate immune checkpoint therapy efficacy.

Conclusion

This study elucidates a microbiota-immune circuit where B. breve-EPS promotes T cell stemness through gut lymphoid tissue education, ultimately enhancing systemic anti-tumor responses. Our findings position microbial EPS as novel mucosal checkpoint modulators, suggesting adjuvant use of B. breve polysaccharides could amplify immune checkpoint blockade efficacy.

Introduction

Wee1 negatively regulates the G2/M checkpoint of the cell cycle, inhibiting cells with DNA damage from entering mitosis and inducing apoptosis. Although efficacy of Wee1 inhibitors as single agents or in combination with other agents has been reported in a variety of carcinomas and the association between Wee1 inhibitors and tumor immunity has been reported, the effect of Wee1 inhibitors on the tumor immune microenvironment in colorectal cancer (CRC) has not been clarified. Since p53 and KRAS are involved in cell cycle regulation at the G1/S checkpoint, mutations in them may increase dependence on the G2/M checkpoint for DNA repair, making Wee1 inhibitors more effective.

Aims & Methods

This study aimed to clarify the association between Wee1 expression and clinicopathological features in CRC and the effect of Wee1 inhibitors on the tumor immune microenvironment in CRC. Study 1: We performed immunostaining on 152 CRC cases surgically resected at our hospital between May 2013 and February 2015, and classified them into positive and negative groups according to the degree of Wee1 expression and investigated the association between Wee1 expression and clinicopathological features. Study 2: We measured Wee1 expression levels by Realtime PCR on 10 human-derived CRC cell lines and 2 mouse-derived CRC cell lines. We examined the effect of Wee1 inhibitor (AZD1775) on cell proliferation using Timelapse system. We examined the effect of Wee1 inhibitor on cell cycle using Cell Cycle Analysis Kit. We examined the effects of Wee1 inhibitor on apoptosis induction were examined using the Apoptosis Detection Kit. Study 3: We created the CRC orthotopic transplantation mouse model by transplanting the CRC cell line MC38 derived from C57BL/6 mice into the cecum of mice, comparing the tumor sizes of the control and treatment groups treated with Wee1 inhibitor for 21 days, and evaluating the effect of Wee1 inhibitor on the tumor immune microenvironment by immunostaining and RNA sequencing of mice tumor specimens.

Results

Study 1: No significant association was found between Wee1 expression and clinicopathological features or prognosis. However, overall survival rate was significantly higher in Wee1 positive and KRAS-mutant patients compared to Wee1 negative and KRAS wild-type patients. Study 2: Real time PCR showed that Wee1 expression was higher in most CRC cell lines than in normal colon mucosa. Wee1 inhibitor attenuated cell proliferation in a concentration-dependent manner in all CRC cell lines, and the effect was independent on Wee1 expression level. Cell cycle analysis showed that the percentage of cells in G2/M phase increased after exposure to Wee1 inhibitor, especially in cell lines with both p53 and KRAS mutations. In addition, the number of apoptotic cells increased after exposure to Wee1 inhibitor, but in the cell lines with KRAS mutation and p53 wild type, the number of apoptotic cells did not increase. Study 3: In the orthotopic transplanted mouse model, tumor volume was significantly smaller in the treated group compared to the control group, and immunohistological examination and RNA sequencing of mice tumor specimens showed enhanced tumor immunity and suppressed stromal reaction.

Conclusion

Wee1 inhibitor attenuated CRC cell proliferation through modulating cell cycle, especially in cell lines with p53 mutation and attenuated tumor growth via enhancement of tumor immune response and suppression of stromal reaction in orthotopic transplanted mouse model. Wee1 inhibitor AZD1775 could be the promising treatment option for CRC.

Introduction

Tumor angiogenesis is considered as a hallmark of cancer¹ and plays a critical role in colorectal cancer (CRC) development and progression^2,3. Tumor vasculature promotes tumor growth and metastasis since it not only provides nutrients and oxygen for the rapid proliferation of cancer cells as well as a route for metastatization, but also affects the infiltration of immune cells to the tumor site. Thus, the targeting of angiogenesis has become an attractive strategy against various human malignancies, including CRC⁴. In fact, tumor-associated vessels are leaky and poorly efficient due to the dismantlement of the basement membrane and the loss of pericytes, thus preventing proficient drug delivery to the tumor⁵. In this context, we demonstrated that Multimerin-2, an extracellular matrix glycoprotein deposited along the blood vessels in juxtaposition between endothelial cells and pericytes, mural cells fundamental in vessels stabilization, is key in the maintenance of vascular homoeostasis⁶. However, the effects of this molecule on pericytes biology and its putative impact on the immune cells’ recruitment are still poorly understood. Since, many vessels associated with CRC lose Multimerin-2 expression, thus we aimed at verifying the effects of this loss in pericyte recruitment, vascular stability, and the surrounding tumor microenvironment.

Aims & Methods

Immunohistochemistry analysis on colorectal cancer patients’ biopsies revealed that colon cancer associated vessels display a downregulated expression of Multimerin-2. Thus, we aimed at verifying the extent of pericyte recruitment by Multimerin-2-devoid vessels in a colon cancer model and at evaluating how Multimerin-2 loss affects the surrounding tumor microenvironment. Human umbilical vein endothelial cells (HUVEC) and Human brain vascular pericytes (HBVP) were used as in vitro models for proliferation, functional assays, transcriptomic and proteomics analyses. Wild type and Multimerin-2^-/-C57BL/6 mice were subcutaneously injected with the syngeneic colon cancer MC-38 cells and pericyte coverage and microenvironmental features were assessed in tumor sections by immunofluorescence as well as drug delivery following cisplatin treatment six hours prior sacrifice.

Results

We demonstrated that Multimerin-2 promotes pericytes migration, adhesion, and proliferation. Consistently, MC-38 xenografts from Multimerin-2 mice displayed a decreased pericytes coverage along the vessels as well as increased intratumoral hypoxia, suggestive of vascular inefficiency. In fact, upon treatments we counted less DNA \cisplatin adducts in tumors from Multimerin-2^-/- mice. Interestingly, tumors from Multimerin-2 mice also displayed an altered CD68 cells infiltrate, suggesting that Multimerin-2 loss can affect macrophages recruitment to the tumor site. Accordingly, a higher number of the monocytic-like cells THP-1 migrated through a Multimerin-2-depleted endothelium. Furthermore, we observed that the loss of Multimerin-2 also associates with the expression of cytokines involved in macrophages recruitment.

Conclusion

These results suggest that Multimerin-2 loss impairs the pericyte recruitment, adhesion and proliferation. As a result, the vasculature lacking Multimerin-2 is utterly destabilized leading to increased intratumoral hypoxia and poorly efficient drug delivery. Importantly, the absence of Multimerin-2 also impairs the immune response suggesting that different Multimerin-2 expression in CRC tumor associated vessels could represent a promising biomarker to predict the efficacy of chemotherapy and/or immune therapy in CRC patient treatment

References

1. Hanahan, D. Hallmarks of Cancer: New Dimensions. Cancer Discovery 12, 31–46 (2022).
2. Weis, S. M. & Cheresh, D. A. Tumor angiogenesis: molecular pathways and therapeutic targets. Nat Med 17, 1359–1370 (2011).
3. Angiogenesis Factors Involved in the Pathogenesis of Colorectal Cancer. Current Health Sciences Journal 5–11 (2013) doi:10.12865/CHSJ.40.01.01.
4. Liu, Z.-L., Chen, H.-H., Zheng, L.-L., Sun, L.-P. & Shi, L. Angiogenic signaling pathways and anti-angiogenic therapy for cancer. Sig Transduct Target Ther 8, 198 (2023).
5. Carmeliet, P. & Jain, R. K. Principles and mechanisms of vessel normalization for cancer and other angiogenic diseases. Nat Rev Drug Discov 10, 417–427 (2011).
6. Pellicani, R. et al. Multimerin-2 maintains vascular stability and permeability. Matrix Biology 87, 11–25 (2020).

Introduction

Colorectal cancer, with an estimated two million new cases each year worldwide, poses a significant socioeconomic burden on modern society. It is the second leading cause of cancer-related deaths, accounting for nearly one million premature deaths annually.

Aims & Methods

The role of Signal Transducer and Activator of Transcription 2 (STAT2) in this context is not yet understood, and no therapies targeting this transcription factor are currently being pursued. We explored the function of STAT2 in colorectal cancer by employing experimental mouse models, as well as conducting functional assays with three-dimensional tumoroids in the context of anticancer therapy.

Results

In vivo studies showed that Stat2KO mice were more resistant to developing tumors in both inflammation-driven and inflammation-independent models of experimental colorectal cancer. This unexpected outcome could not be reconciled with the known role of STAT2 in inducing cell death downstream of type I interferon signaling. We consequently performed ex vivo experiments with tumoroids derived from Stat2KO mice carrying the multiple intestinal neoplasia (Min) mutant allele of the adenomatous polyposis coli (Apc) gene. These tumoroids exhibited slower growth, were generally smaller in size, and showed a higher degree of differentiation compared to those derived from ApcMin/+ wildtype mice. Remarkably, when treated with anti-tumor agents that induce cell death through various mechanisms, tumoroids derived from ApcMin/+ Stat2KO mice were found to be highly sensitive. Additional functional studies indicated that the underlying mechanism might involve a STAT2-dependent disruption of innate immune DNA sensing.

Conclusion

Our results establish STAT2 as a promoter of colorectal cancer and a contributor to resistance against anti-tumor therapy. These findings suggest that targeting STAT2-dependent signaling could be a promising therapeutic strategy for patients with colorectal cancer.

Introduction

Since currently utilized screening tools including fecal immunochemical test and serum tumor markers cannot reveal sufficient diagnostic powers for colorectal cancer (CRC) due to their low sensitivities. It is thus hopeful to discover non-invasive diagnostic biomarkers for CRC. Urine is one of the most attractive samples for mass-screening because of the non-invasiveness and easy handling. We have previously reported many types of urinary biomarkers for gastrointestinal malignancies.

Aims & Methods

We conducted the current study to discover and establish urinary protein biomarkers that enables early detection of CRC. Among 475 patients including 299 healthy controls (HCs) and 175 stage 0-III CRC patients, age- and sex-matched 373 patients were enrolled in this case-control study. A whole cohort was randomly divided into the discovery cohort (n=32), training cohort (n=220) and validation cohort (n=111). In the discovery cohort, we have performed the isobaric tags for relative and absolute quantitation (iTRAQ) -coupled liquid chromatography-mass spectrometry (LC-MS) to identify urinary protein biomarker candidates. After that, the urinary protein biomarkers were established with ELISA in the training cohort. Finally, the established biomarkers were validated with ELISA in the independent validation cohort.

Results

In the discovery cohort, we identified 78 urinary proteins with aberrant expression between HCs and CRC patients. Among them, urinary levels of 11 proteins were significantly higher in the CRC patients than in the HCs, and 4 urinary proteins were independent biomarkers on the multivariate analysis in the training cohort. In the validation cohort, these urinary proteins also showed significantly higher levels in the CRC patients than in the HCs. Of these urinary biomarkers, DPEP1 and TFF1 were top two urinary protein biomarkers for the diagnosis of CRC, and the combination biomarker panel using these two proteins showed an excellent AUC of 0.870 with 94.3% sensitivity and 63.4% specificity for the diagnosis of stage 0-III CRC. Notably, this combination biomarker panel could distinguish stage 0-I CRC that are curable with endoscopic resection, with an AUC of 0.852, 93.8% sensitivity and 61.6% specificity.

Conclusion

The established novel urinary biomarker panel provides a promising method for early detection of CRC with completely non-invasive manner.

Introduction

Colorectal cancer (CRC) is the third-most common malignancy worldwide. Lysyl oxidase-like protein 2 (LOXL2), which promotes crosslinking of collagen and elastin, contributes to tumor progression in different tumour entities, but its role in CRC has not been evaluated. Here, we unraveled the role of LOXL2 in regulating tumour immune microenvironment of CRC and identified LOXL2 as a potential target for immunotherapy.

Aims & Methods

We analyzed the expression of LOXL2 in the Cancer Genome Atlas (TCGA) data. The effect of LOXL2 on CRC cell growth was determined by cell viability and colony formation assays. In vivo models were established by subcutaneous injection of colorectal cancer cell lines into immunodeficient NOD-SCID-γ (NSG) mice and immunocompetent wild-type BALB/c mice. Flow cytometry and immunohistochemistry were used to analyze immune cell composition. The collagen fibers in the extracellular matrix (ECM) were observed by masson staining, second harmonic generation imaging (SHG), and two-photon fluorescence imaging.

Results

Survival prognosis analysis revealed that high LOXL2 expression was correlated with poor survival of patients with CRC in TCGA cohort. However, we found that LOXL2 overexpression in human (HCT116 and DLD1) and mouse (CT26 and MC38) CRC cell lines had little effect on tumor growth in vitro and in immunodeficient mice. But its overexpression in mouse CRC cell lines could significantly promote tumor growth in immunocompetent mice (p<0.05). Overexpression of LOXL2 led to CXCL1 upregulation in the supernatant of CRC cells and CRC tumor tissues; subsequently, upregulation of CXCL1 induced more migration of myeloid-derived suppressor cells (MDSCs) in vitro and recruited more MDSCs in the tumor microenvironment. On the other hand, LOXL2 increased the crosslinking of collagen fibers in vitro and in the tumor microenvironment, which subsequently activated the function of MDSCs (up-expression of Arg1, IDO1, and INOS) and increased the oncostatin M (OSM) secreted by MDSCs. In turn, OSM secreted by MDSCs induced the expression of LOXL2 in CRC cells. The accumulation and activation of MDSCs in the tumor microenvironment inhibited the infiltration and function of CD8+ T cells, which suppressed the anti-tumour immunity of the host. Targeting of LOXL2 with a neutralizing antibody or inhibitor (PAT-1251) significantly impaired tumor growth (p<0.05) and MDSC infiltration and activation (p<0.05) in immunodeficient mice. Finally, PAT-1251 boosted anti-PD1 efficacy in colorectal cancer, suggesting that it could be repurposed to improve immunotherapy efficacy.

Conclusion

Our findings revealed that LOXL2 exerted a pro-tumorigenic role in CRC. LOXL2 recruited and activated MDSCs through CXCL1 and crosslinking of collagen fibers, respectively, subsequently reducing infiltration and activation of anti-tumor CD8⁺ T cells in the tumor microenvironment. Targeting this axis with PAT-1251 improved the efficacy of immunotherapy in colorectal cancer.