Introduction

The MYC oncogene plays a pivotal role in variable cellular functions including proliferation, differentiation, metabolism, protein synthesis, survival and apoptosis, while it is also considered a crucial regulator of tumor immune microenvironment.

Aims & Methods

The purpose of our research is the evaluation of the immunohistochemical expression of MYC protein in primary colorectal adenocarcinomas and its correlation with prognosis, as well as with the qualitative and quantitative characteristics of the immune microenvironment.
In our study, a total of 94 patients with colorectal adenocarcinoma were included [59 men/35 women, median age (IQR): 71 (44-88) years, I-IV stage (AJCC/TNM 8^th edition)] who underwent surgical resection between January 2004 to May 2008. Histological slides and paraffin blocks were retrieved from the archives of the Department of Pathology, Attikon University Hospital. Tissue microarrays (TMAs) of 2 mm diameter were constructed with representative areas from the center and the periphery of the tumor. Immunohistochemical stains were performed on TMAs to detect the expression of MYC and Ki67 (proliferation index) by neoplastic cells and the presence of adaptive immunity (CD3, CD4, CD8, CD20) cells. MYC immunostaining in >20% of neoplastic cells was scored as positive (over-expressed). Clinical and pathologic information was acquired from hospital medical records including patient’s outcome and survival.

Results

MYC over-expression (>20%) in the center and periphery of the tumor was observed in 59 patients (63%) and is significantly associated with tumor infiltration by increased number of CD3+, CD4+, CD8+ T and CD20+ B lymphocytes as well as with an increased index of cell proliferation Ki-67 (p<0.001). In addition, positive MYC expression in the tumor periphery is significantly correlated with longer overall survival compared to cases with weak MYC expression (70 vs 48,5 months; p=0.046; p<0.05).

Conclusion

In patients with colorectal adenocarcinoma, increased MYC expression by neoplastic cells is associated with increased densities of adaptive immune cells in the tumor microenvironment. In addition, it is associated with a favorable prognosis, possibly through a more effective antitumor immune response. Consequently, the expression of MYC might be a useful predicting indicator of anti-tumor immunity status and patient’s prognosis.

References

1. Dhanasekaran et al. The MYC oncogene - the grand orchestrator of cancer growth and immune evasion. Nat Rev Clin Oncol. 2022 Jan;19(1):23-36.
2. Lee et al. Favorable prognosis in colorectal cancer patients with co-expression of c-MYC and ß-catenin. BMC Cancer. 2016 2016 Sep 13;16(1):730.

Introduction

Although the prognostic significance of cellular components of the immune system has been investigated in several solid neoplasms and is well established, the role of plasma cells in the tumor microenvironment has been neglected and only recently these cells are increasingly being appreciated as key players in the tumor microenvironment.

Aims & Methods

The purpose of our study is to explore the contribution of plasma cells to the immune microenvironment of colorectal adenocarcinomas and describe their correlation with prognosis.
In our study, a total of 108 patients with colorectal adenocarcinoma were included [68 men/40 women, median age (IQR): 70 (64-76) years, I-IV stage (AJCC/TNM 8^th edition)] who underwent surgical resection between January 2004 to May 2008. Histological slides and paraffin blocks were retrieved from the archives of the Department of Pathology, Attikon University Hospital. Tissue microarrays (TMAs) of 2 mm diameter were constructed with representative areas from the center and periphery of the tumor. Immunohistochemical stains were performed on TMAs to detect the presence of plasma cells (CD138) and cells of adaptive immunity (CD3, CD4, CD8, CD20). Tertiary lymphoid structures (TLS) were assessed on hematoxylin-eosin-stained slides. Depending on the number of plasma cells, the cases were classified into the following categories: absence of plasma cells or presence of rare single cells, presence of rare small aggregates (>5 cells/aggregation), presence of frequent aggregates, presence of large aggregates or sheets of plasma cells. Clinical and pathologic information was acquired from hospital medical records including patient’s outcome and survival.

Results

Ιncreased number of plasma cells (presence of frequent aggregates, presence of large aggregates or sheets of plasma cells) was observed in 70 cases (65%), while absence or rare aggregates of plasma cells was demonstrated 38 cases (35 %). The presence of large aggregates or sheets of plasma cells in the tumor was significantly associated with the presence of TLS (p=0.004) and tumor infiltration by increased numbers of CD3⁺, CD8⁺ T, and CD20⁺ B lymphocytes compared with the absence of plasma cells or the presence of rare single plasma cells (p=0.025, p=0.012, p=0.001, respectively). Moreover, it was reported that the presence of an increased number of plasma cells compared with the absence of plasma cells in the center of the tumor is significantly correlated to a more favorable prognosis (77 vs 47 months; p=0.047; p<0.05).

Conclusion

In patients with colorectal adenocarcinoma, the presence of high densities of plasma cells in the tumor bed may be associated with increased adaptive immune cell populations in the tumor environment, as well as with longer patient’s survival. Consequently, a careful evaluation of plasma cells and a better understanding of their role in the tumor microenvironment of colorectal cancer seems to be crucial in order to design efficient immunotherapeutic strategies.

References

1.Galon et al. Tumor immunology and tumor evolution: intertwined hisories. Immunity. 2020 Jan 14;52(1):55-81.
2.Hegde et al. Top 10 Challenges in Cancer Immunotherapy. Immunity. 2020 Jan 14;52(1):17-35.

Introduction

Immune checkpoint blockade (ICB) for treatment of colorectal cancer (CRC) has been approved for clinical use since 2017. However, the approval of PD-L1 inhibitor for CRC has been encumbered by the lack of durable clinical benefits. Increasing evidence from other cancer types support that PD-L1 expression correlates to the efficacy of ICB and tumor PD-L1 is essential for ICB-mediated tumor cytotoxicity [1-3]. Concomitantly, many studies have also demonstrated the infrequent expression of PD-L1 in CRC cells [4, 5], thereby alluding to the possibility of low/absent PD-L1 expression as a primary obstacle to ICB efficacy.

Aims & Methods

Given the integral role of tumour microenvironment (TME) in shaping the tumour’s natural history, we sought to elucidate the role of tumour stroma in regulating CRC PD-L1 expression. To this end, we aim to rescue tumor PD-L1 and assess its utility in ICB-mediated anti-tumour immunity.
​To explore the role of different cell types in the TME on CRC PD-L1 expression, cocultures between major cell compartments of the TME and CRC cell lines were performed. Further various omics approaches were utilised to characterise the crosstalk between CRC and stromal cells that saw significant suppression of CRC PD-L1 expression.

Results

Myeloid cells are able to exert a significant suppression of PD-L1 expression in CRC and this suppression is regulated at a post-transcriptional level. The underlying mechanism is orchestrated by CRC metabolic shift from glycolysis to oxidative phosphorylation in response to harsh metabolic competition within the TME.

Conclusion

We have identified potential cellular compartment of the TME and mechanisms which may explain, in part, low/absent CRC PD-L1 expression. This can thus serve to evaluate appropriate external manipulations that can alleviate CRC PD-L1 suppression and explore the possibility of combinatory therapy with anti-PD-L1 ICB to improve its clinical efficacy and durability.

References

1. Herbst, R.S., et al., Atezolizumab for First-Line Treatment of PD-L1–Selected Patients with NSCLC. New England Journal of Medicine, 2020. 383(14): p. 1328-1339.
2. Cortes, J., et al., Pembrolizumab plus Chemotherapy in Advanced Triple-Negative Breast Cancer. New England Journal of Medicine, 2022. 387(3): p. 217-226.
3. Lau, J., et al., Tumour and host cell PD-L1 is required to mediate suppression of anti-tumour immunity in mice. Nature Communications, 2017. 8(1): p. 14572.
4. Llosa, N.J., et al., The Vigorous Immune Microenvironment of Microsatellite Instable Colon Cancer Is Balanced by Multiple Counter-Inhibitory Checkpoints. Cancer Discovery, 2015. 5(1): p. 43-51.
5. Taube, J.M., et al., Association of PD-1, PD-1 Ligands, and Other Features of the Tumor Immune Microenvironment with Response to Anti–PD-1 Therapy. Clinical Cancer Research, 2014. 20(19): p. 5064-5074.

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

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(CRC) is the third most common cancer in the world and the fourth most common cause of cancer death. Its incidence has continued to increase in recent years. MicroRNAs (miRNAs) are the most studied non-coding RNAs. In the process of tumorigenesis and development, miRNA plays an important role as an inhibitor or proto-oncogene of tumors, which makes miRNAs extremely attractive targets for new treatment methods of tumors. microRNA-derived protein (MidP619) is highly specific and highly expressed in most tumors, but expressed low in normal cells. The downregulation of MidP619 hinders the proliferation and metastasis of various tumor cell lines. The results of this study reveal for the first time the important role of miRNA at the 3' end, confirm a novel protein translation mode, and elucidate its important role in tumorigenesis and development through relevant experiments.

Aims & Methods

The expression of MidP619 in CRC and non-cancerous tissue was detected by immunohistochemical staining. CO-IP and protein mass spectrometry were used to identify MidP619 and its associated interaction proteins, and bioinformatics analysis was performed on their interaction protein data. After overexpression of MidP619 by protein mass spectrometry analysis and proteomics-related bioinformatics, the relevant protein components and their changes were studied. Transwell assay was used to study tumor invasion and migration; CCK-8 analysis to study tumor metabolic capacity; Edu was used to analyze tumor cell proliferation; Tumor migration ability was detected by wound healing assay; Colony formation experiments were used to identify tumor colony formation. A series of in vivo experiments were conducted to help reveal the mechanisms of MidP169 in colorectal cancer.

Results

MidP169 expression was markedly higher in CRC tissues than in non-tumour tissues. Through protein interaction and protein mass spectrometry, the interaction between MidP619 and angiogenesis-related proteins ANXA2, ANXA1 and PARVA was verified. The MidP619 overexpression in the colorectal cancer cell line was found that it had an important role in the occurrence and development of tumors through proteomic verification. Through overexpression and knockout of MidP619, Transwell, CCK-8, Edu, wound healing and colony formation experiments, it was verified that MidP619 plays an important role in the proliferation, migration and invasion ability of CRC cells. Western blot experiments preliminarily verified that MidP619 may play an important role in tumor epithelial-mesenchymal transformation (EMT) or tumor angiogenesis. Finally, overexpression of MidP169 increased the malignancy of the tumor and angiogenesis-related treatments inhibited tumour growth in a xenograft mouse model.

Conclusion

Through the study of MidP619 in CRC, it has been confirmed that MidP619 is widely involved in the proliferation, invasion, migration, metabolism, angiogenesis and other factors leading to increased malignancy of colorectal cancer at the cellular, animal and clinical levels. Through interaction proteins and proteomics experiments, the interacting proteins of MidP619 and the related pathways involved in them were discovered. It provides more evidence for the protein translated by the new model of MidP to play a role in the body, especially tumors.