Differential landscape of immune evasion in oncogenic RAS-driven primary and metastatic colorectal cancers.

Oncogenic drivers such as KRAS extensively modulate the tumor inflammatory microenvironment (TIME) of colorectal cancer (CRC). The influence of KRAS on modulating immune cell composition remains unclear. The objective of this study was to identify signatures of infiltrative immune cells and distinctive patterns that differ between RAS wild-type (WT) and oncogenic mutant (MT) CRC that explain immune evasion in MT tumors. A total of 7,801 CRC specimens were analyzed using next-generation DNA sequencing, whole-exome sequencing, and/or whole transcriptome sequencing. Deficiency of mismatch repair (dMMR)/microsatellite instability (MSI) and tumor mutation burden (TMB) were also assessed. KRAS mutations were present in 48% of CRC, similarly distributed in patients younger than vs. 50 years and older. In microsatellite stable (MSS) KRAS MT tumors, composition of the TIME included higher neutrophil infiltration and lower infiltration of B cells. MSI-H/dMMR was significantly more prevalent in RAS WT (9.1%) than in KRAS MT (2.9%) CRC. In MSS CRC, TMB-high cases were significantly higher in RAS MT (3.1%) than in RAS WT (2.1%) tumors. KRAS and NRAS mutations are associated with increased neutrophil infiltration, with codon-specific differences. These results demonstrate significant differences in the TIME of RAS mutant CRC that match previous reports of immunoevasive characteristics of such tumors.

Centrosomal Protein 55 (CEP55) Drives Immune Exclusion and Resistance to Immune Checkpoint Inhibitors in Colorectal Cancer.

Colorectal cancer (CRC) currently ranks as the third most common cancer in the United States, and its incidence is on the rise, especially among younger individuals. Despite the remarkable success of immune checkpoint inhibitors (ICIs) in various cancers, most CRC patients fail to respond due to intrinsic resistance mechanisms. While microsatellite instability-high phenotypes serve as a reliable positive predictive biomarker for ICI treatment, the majority of CRC patients with microsatellite-stable (MSS) tumors remain ineligible for this therapeutic approach. In this study, we investigated the role of centrosomal protein 55 (CEP55) in shaping the tumor immune microenvironment in CRC. CEP55 is overexpressed in multiple cancer types and was shown to promote tumorigenesis by upregulating the PI3K/AKT pathway. Our data revealed that elevated CEP55 expression in CRC was associated with reduced T cell infiltration, contributing to immune exclusion. As CRC tumors progressed, CEP55 expression increased alongside sequential mutations in crucial driver genes (APC, KRAS, TP53, and SMAD4), indicating its involvement in tumor progression. CEP55 knockout significantly impaired tumor growth in vitro and in vivo, suggesting that CEP55 plays a crucial role in tumorigenesis. Furthermore, the CEP55 knockout increased CD8+ T cell infiltration and granzyme B production, indicating improved anti-tumor immunity. Additionally, we observed reduced regulatory T cell infiltration in CEP55 knockout tumors, suggesting diminished immune suppression. Most significantly, CEP55 knockout tumors demonstrated enhanced responsiveness to immune checkpoint inhibition in a clinically relevant orthotopic CRC model. Treatment with anti-PD1 significantly reduced tumor growth in CEP55 knockout tumors compared to control tumors, suggesting that inhibiting CEP55 could improve the efficacy of ICIs. Collectively, our study underscores the crucial role of CEP55 in driving immune exclusion and resistance to ICIs in CRC. Targeting CEP55 emerges as a promising therapeutic strategy to sensitize CRC to immune checkpoint inhibition, thereby improving survival outcomes for CRC patients.

Allogeneic tumor cell-derived extracellular vesicles stimulate CD8 T cell response in colorectal cancer.

Most colorectal cancer (CRC) patients present with a microsatellite-stable phenotype, rendering them resistant to immune checkpoint inhibitors (ICIs). Among the contributors to ICI resistance, tumor-derived extracellular vesicles (TEVs) have emerged as critical players. Previously we demonstrated that autologous transfer of TEVs without miR-424 can induce tumor antigen-specific immune responses in CRC models. Therefore, we postulated that allogeneic TEVs, modified to lack miR-424 and derived from an MC38 cells, could induce CD8+ T cell responses while restraining CT26 cell-based tumor. Here, we show that prophylactic administration of MC38 TEVs, without miR-424, showed a significant augmentation in CD8+ T-cells within CT26 tumors. This allogenic TEV effect was evident in CT26 tumors but not B16-F10 melanoma. Furthermore, we demonstrated the capacity of dendritic cells (DCs) to internalize TEVs, a possible mechanism to elicit immune response. Our investigation of autologously administered DCs, which had been exposed to modified TEVs, underscores their potential to dampen tumor growth while elevating CD8+ T cell levels vis-a-vis MC38 wild-type TEVs exposed to DCs. Notably, the modified TEVs were well tolerated and did not increase peripheral blood cytokine levels. Our findings underscore the potential of modified allogeneic TEVs without immune-suppressive factors to elicit robust T cell responses and limit tumor growth.

Advancing Cancer Immunotherapy: From Molecular Mechanisms to Clinical Applications.

In recent years, cancer immunotherapy research has made remarkable progress, completely transforming the cancer treatment landscape [...].

Distinct mechanisms of PTEN inactivation in dogs and humans highlight convergent molecular events that drive cell division in the pathogenesis of osteosarcoma.

A hallmark of osteosarcoma in both human and canine tumors is somatic fragmentation and rearrangement of chromosome structure which leads to recurrent increases and decreases in DNA copy number. The PTEN gene has been implicated as an important tumor suppressor in osteosarcoma via forward genetic screens. Here, we analyzed copy number changes, promoter methylation and transcriptomes to better understand the role of PTEN in canine and human osteosarcoma. Reduction in PTEN copy number was observed in 23 of 95 (25%) of the canine tumors examined leading to corresponding decreases in PTEN transcript levels from RNA-Seq samples. Unexpectedly, canine tumors with an intact PTEN locus had higher levels of PTEN transcripts than human tumors. This variation in transcript abundance was used to evaluate the role of PTEN in osteosarcoma biology. Decreased PTEN copy number and transcript level was observed in - and likely an important driver of - increases in cell cycle transcripts in four independent canine transcriptional datasets. In human osteosarcoma, homozygous copy number loss was not observed, instead increased methylation of the PTEN promoter was associated with increased cell cycle transcripts. Somatic modification of PTEN, either by homozygous deletion in dogs or by promoter methylation in humans, is clinically relevant to osteosarcoma, because the cell cycle related transcripts are associated with patient outcomes. The PTEN gene is part of a syntenic rearrangement unique to the canine genome, making it susceptible to somatic loss of both copies of distal chromosome 26 which also includes the FAS death receptor. SIGNIFICANCE STATEMENT: PTEN function is abrogated by different mechanisms in canine and human osteosarcoma tumors leading to uncontrolled cell cycling. Somatic loss of this canine specific syntenic region may help explain why the canine genome appears to be uniquely susceptible to osteosarcoma. Syntenic arrangement, in the context of copy number change, may lead to synergistic interactions that in turn modify species specific cancer risk. Comparative models of tumorigenesis may utilize different driver mechanisms.

Allogeneic Tumor Cell-Derived Extracellular Vesicles Stimulate CD8 T Cell Response in Colorectal Cancer.

Colorectal Cancer (CRC) is the second leading cause of cancer-related death in the United States. Most CRC patients present with a microsatellite stable (MSS) phenotype and are highly resistant to immunotherapies. Tumor extracellular vesicles (TEVs), secreted by tumor cells, can contribute to intrinsic resistance to immunotherapy in CRC. We previously showed that autologous TEVs without functional miR-424 induce anti-tumor immune responses. We hypothesized that allogeneic modified CRC-TEVs without miR-424 (mouse homolog miR-322) derived from an MC38 background would effectively stimulate CD8+ T cell response and limit CT26 tumor growth. Here we show that prophylactic administration of MC38 TEVs without functional miR-424 significantly increased CD8+ T cells in CT26 CRC tumors and limited tumor growth, not B16-F10 melanoma tumors. We further show that the depletion of CD4+ and CD8+ T cells abolished the protective effects of MC38 TEVs without functional miR-424. We further show that TEVs can be taken up by DCs in vitro, and subsequent prophylactic administration of autologous DCs exposed to MC38 TEVs without functional miR-424 suppressed tumor growth and increased CD8+ T cells compared to MC38 wild-type TEVs exposed to DCs, in Balb/c mice bearing CT26 tumors. Notably, the modified EVs were well tolerated and did not increase cytokine expression in peripheral blood. These findings suggest that allogeneic-modified CRC-EVs without immune suppressive miR-424 can induce antitumor CD8+ T cell responses and limit tumor growth in vivo.

Fecal Microbiota Restoration Modulates the Microbiome in Inflammation-Driven Colorectal Cancer.

Chronic inflammation of the colon (colitis) is a known risk factor for inflammatory-driven colorectal cancers (id-CRCs), and intestinal microbiota has been implicated in the etiology of id-CRCs. Manipulation of the microbiome is a clinically viable therapeutic approach to limiting id-CRCs. To understand the microbiome changes that occur over time in id-CRCs, we used a mouse model of id-CRCs with the treatment of azoxymethane (AOM) and dextran sodium sulfate (DSS) and measured the microbiome over time. We included cohorts where the microbiome was restored using cage bedding swapping and where the microbiome was depleted using antibiotics to compare to untreated animals. We identified consistent increases in Akkermansia in mice receiving horizontal microbiome transfer (HMT) via cage bedding swapping, while the control cohort had consistent longitudinal increases in Anaeroplasma and Alistipes. Additionally, fecal lipocalin-2 (Lcn-2), a marker of intestinal inflammation, was elevated in unrestored animals compared to restored and antibiotic-treated counterparts following HMT. These observations suggest a potential role for Akkermansia, Anaeroplasma, and Alistipes in regulating colonic inflammation in id-CRCs.

Tumor-derived extracellular vesicles in the colorectal cancer immune environment and immunotherapy.

Despite significant advances in the screening, diagnosis, and treatment of colorectal cancer (CRC) immune checkpoint inhibitors (ICIs) continue to have limited utility outside of microsatellite-high disease. Given the durable response to immunotherapy seen across malignancies, increasing CRC response rates to ICI therapy is an active area of clinical research. An increasing body of work has demonstrated that tumor-derived extracellular vesicles (TEVs) are key modulators in tumor signaling and the determinants of the tumor microenvironment. Pre-clinical models have shown that TEVs are directly involved in antigen presentation and are involved in radiation-induced DNA damage signaling. Both direct and indirect modifications of these TEVs can alter CRC immunogenicity and ICI treatment response, making them attractive targets for potential therapeutic development. In addition, modified TEVs can be developed using several different mechanisms, with varied cargo including micro-RNAs and small peptide molecules. Recent work has shown strong pre-clinical evidence of injected modified TEV-induced ICI activity, with knockdown of the micro-RNA miR-424 in TEVs improving CRC immunogenicity and increasing anti-PD-1 activity in mouse models. Clinical trials are ongoing in the evaluation of modified TEVs in cancer therapy, but they appear to be a promising therapeutic target in CRC.

Gaucher disease - more than just a rare lipid storage disease.

Gaucher disease (GD), one of the most common lysosomal storage diseases, is caused by mutations in the gene, GBA1, that leads to defective glucocerebrosidase activity resulting in the accumulation and storage of glycosphingolipids. However, the pathophysiology of GD is more complicated leading to various associated conditions such as skeletal manifestations and Parkinson's disease (PD). These may result from oxidative stress and inflammatory responses due to complex interconnection of downstream factors such as substrate accumulation, endoplasmic reticulum (ER) stress, unfolded protein response (UPR), calcium dysregulation, mitochondrial dysfunction, defective autophagy, accumulation of α-synuclein aggregates, altered secretion and function of extracellular vesicles (EVs), and immunologic hyperactivity. Here we provide an overview of lysosomal storage diseases followed by a comprehensive review of the factors contributing to oxidative stress and inflammation in GD pathophysiology, mechanisms underlying the possible associated complications, current established treatments for GD, their limitations, and potential primary and adjunctive treatment options targeting these factors.

Comparative analysis of genome-wide DNA methylation identifies patterns that associate with conserved transcriptional programs in osteosarcoma.

Osteosarcoma is an aggressive tumor of the bone that primarily affects young adults and adolescents. Osteosarcoma is characterized by genomic chaos and heterogeneity. While inactivation of tumor protein p53 (TP53) is nearly universal other high frequency mutations or structural variations have not been identified. Despite this genomic heterogeneity, key conserved transcriptional programs associated with survival have been identified across human, canine and induced murine osteosarcoma. The epigenomic landscape, including DNA methylation, plays a key role in establishing transcriptional programs in all cell types. The role of epigenetic dysregulation has been studied in a variety of cancers but has yet to be explored at scale in osteosarcoma. Here we examined genome-wide DNA methylation patterns in 24 human and 44 canine osteosarcoma samples identifying groups of highly correlated DNA methylation marks in human and canine osteosarcoma samples. We also link specific DNA methylation patterns to key transcriptional programs in both human and canine osteosarcoma. Building on previous work, we built a DNA methylation-based measure for the presence and abundance of various immune cell types in osteosarcoma. Finally, we determined that the underlying state of the tumor, and not changes in cell composition, were the main driver of differences in DNA methylation across the human and canine samples. SIGNIFICANCE: Genome wide comparison of DNA methylation patterns in osteosarcoma across two species lays the ground work for the exploration of DNA methylation programs that help establish conserved transcriptional programs in the context of varied mutational landscapes.

Tumor models to assess immune response and tumor-microbiome interactions in colorectal cancer.

Despite significant advances over the past 2 decades in preventive screening and therapy aimed at improving patient survival, colorectal cancer (CRC) remains the second most common cause of cancer death in the United States. The average 5-year survival rate of CRC patients with positive regional lymph nodes is only 40%, while less than 5% of patients with distant metastases survive beyond 5 years. There is a critical need to develop novel therapies that can improve overall survival in patients with poor prognoses, particularly since 60% of them are diagnosed at an advanced stage. Pertinently, immune checkpoint blockade therapy has dramatically changed how we treat CRC patients with microsatellite-instable high tumors. Furthermore, accumulating evidence shows that changes in gut microbiota are associated with the regulation of host antitumor immune response and cancer progression. Appropriate animal models are essential to deciphering the complex mechanisms of host antitumor immune response and tumor-gut microbiome metabolic interactions. Here, we discuss various mouse models of colorectal cancer that are developed to address key questions on tumor immune response and tumor-microbiota interactions. These CRC models will also serve as resourceful tools for effective preclinical studies.

Cancer Immunology and Immunotherapies: Mechanisms That Affect Antitumor Immune Response and Treatment Resistance.

The past decade has seen immunotherapy rise to the forefront of cancer treatment [...].

ACKR4 in Tumor Cells Regulates Dendritic Cell Migration to Tumor-Draining Lymph Nodes and T-Cell Priming.

Colorectal cancer (CRC) is one of the most common malignancies in both morbidity and mortality. Immune checkpoint blockade (ICB) treatments have been successful in a portion of mismatch repair-deficient (dMMR) CRC patients but have failed in mismatch repair-proficient (pMMR) CRC patients. Atypical Chemokine Receptor 4 (ACKR4) is implicated in regulating dendritic cell (DC) migration. However, the roles of ACKR4 in CRC development and anti-tumor immunoregulation are not known. By analyzing human CRC tissues, transgenic animals, and genetically modified CRC cells lines, our study revealed an important function of ACKR4 in maintaining CRC immune response. Loss of ACKR4 in CRC is associated with poor immune infiltration in the tumor microenvironment. More importantly, loss of ACKR4 in CRC tumor cells, rather than stromal cells, restrains the DC migration and antigen presentation to the tumor-draining lymph nodes (TdLNs). Moreover, tumors with ACKR4 knockdown become less sensitive to immune checkpoint blockade. Finally, we identified that microRNA miR-552 negatively regulates ACKR4 expression in human CRC. Taken together, our studies identified a novel and crucial mechanism for the maintenance of the DC-mediated T-cell priming in the TdLNs. These new findings demonstrate a novel mechanism leading to immunosuppression and ICB treatment resistance in CRC.

Development of an exosomal gene signature to detect residual disease in dogs with osteosarcoma using a novel xenograft platform and machine learning.

Osteosarcoma has a guarded prognosis. A major hurdle in developing more effective osteosarcoma therapies is the lack of disease-specific biomarkers to predict risk, prognosis, or therapeutic response. Exosomes are secreted extracellular microvesicles emerging as powerful diagnostic tools. However, their clinical application is precluded by challenges in identifying disease-associated cargo from the vastly larger background of normal exosome cargo. We developed a method using canine osteosarcoma in mouse xenografts to distinguish tumor-derived from host-response exosomal messenger RNAs (mRNAs). The model allows for the identification of canine osteosarcoma-specific gene signatures by RNA sequencing and a species-differentiating bioinformatics pipeline. An osteosarcoma-associated signature consisting of five gene transcripts (SKA2, NEU1, PAF1, PSMG2, and NOB1) was validated in dogs with spontaneous osteosarcoma by real-time quantitative reverse transcription PCR (qRT-PCR), while a machine learning model assigned dogs into healthy or disease groups. Serum/plasma exosomes were isolated from 53 dogs in distinct clinical groups ("healthy", "osteosarcoma", "other bone tumor", or "non-neoplastic disease"). Pre-treatment samples from osteosarcoma cases were used as the training set, and a validation set from post-treatment samples was used for testing, classifying as "osteosarcoma detected" or "osteosarcoma-NOT detected". Dogs in a validation set whose post-treatment samples were classified as "osteosarcoma-NOT detected" had longer remissions, up to 15 months after treatment. In conclusion, we identified a gene signature predictive of molecular remissions with potential applications in the early detection and minimal residual disease settings. These results provide proof of concept for our discovery platform and its utilization in future studies to inform cancer risk, diagnosis, prognosis, and therapeutic response.

Tumor-Secreted Extracellular Vesicles Regulate T-Cell Costimulation and Can Be Manipulated To Induce Tumor-Specific T-Cell Responses.

BACKGROUND & AIMS: Colorectal cancer is a major cause of cancer-related deaths worldwide. Immune checkpoint blockade therapies are effective in 30%-60% of the microsatellite instable-high subtype. Unfortunately, most patients with colorectal cancer (>85%) have microsatellite stable tumors that do not respond. In this study, we aimed to decipher the underlying tumor-intrinsic mechanisms critical for improving immunotherapy in colorectal cancer. METHODS: We used human and mouse tumor samples, cell lines, human colorectal cancer organoids, and various syngeneic orthotopic mouse models of late-stage colorectal cancer to define the effects of tumor cell-secreted extracellular vesicles (EVs) on antitumor immune response. RESULTS: Our analyses of human colorectal cancer immune profiles and tumor-immune cell interactions showed that tumor-secreted EVs containing microRNA miR-424 suppressed the CD28-CD80/86 costimulatory pathway in tumor-infiltrating T cells and dendritic cells, leading to immune checkpoint blockade resistance. Modified tumor-secreted EVs with miR-424 knocked down enhanced T-cell-mediated antitumor immune response in colorectal cancer tumor models and increased the immune checkpoint blockade response. Intravenous injections of modified tumor-secreted EVs induced tumor antigen-specific immune responses and boosted the immune checkpoint blockade efficacy in colorectal cancer models that mimic aggressively progressing, late-stage disease. CONCLUSIONS: Collectively, we show a critical role for tumor-secreted EVs in antitumor immune regulation and immunotherapy response, which could be developed as a novel treatment for immune checkpoint blockade-resistant colorectal cancer.

MicroRNA-155 contributes to plexiform neurofibroma growth downstream of MEK.

MicroRNAs (miRs) are small non-coding RNAs that can have large impacts on oncogenic pathways. Possible functions of dysregulated miRs have not been studied in neurofibromatosis type 1 (NF1) plexiform neurofibromas (PNFs). In PNFs, Schwann cells (SCs) have biallelic NF1 mutations necessary for tumorigenesis. We analyzed a miR microarray comparing with normal and PNF SCs and identified differences in miR expression, and we validated in mouse PNFs versus normal mouse SCs by qRT-PCR. Among these, miR-155 was a top overexpressed miR, and its expression was regulated by RAS/MAPK signaling. Overexpression of miR-155 increased mature Nf1-/- mouse SC proliferation. In SC precursors, which model tumor-initiating cells, pharmacological and genetic inhibition of miR-155 decreased PNF-derived sphere numbers in vitro, and we identified Maf as a miR-155 target. In vivo, global deletion of miR-155 significantly decreased tumor number and volume, increasing mouse survival. Fluorescent nanoparticles entered PNFs, suggesting that an anti-miR might have therapeutic potential. However, treatment of established PNFs using anti-miR-155 peptide nucleic acid-loaded nanoparticles marginally decreased tumor numbers and did not reduce tumor growth. These results suggest that miR-155 plays a functional role in PNF growth and/or SC proliferation, and that targeting neurofibroma miRs is feasible, and might provide novel therapeutic opportunities.

Intestinal organoids: a model to study the role of microbiota in the colonic tumor microenvironment.

Colorectal cancer (CRC) is the third most common cause of cancer worldwide. Recent studies have suggested that a dysbiotic shift in the intestinal microbial composition of CRC patients influences tumorigenesis. Gut microbes are known to be integral for intestinal homeostasis; however, the mechanisms by which they impact CRC are unclear. Further knowledge about these complex interactions may guide future CRC management. Thus, it is crucial to establish high-quality experimental models to understand the relationship between host, tumor, microbiota and their metabolic interactions. In this review, we highlight the significance of intestinal microbiota and their metabolites in CRC, challenges with current experimental models, advantages and limitations of organoid culture and future directions of this novel model system in CRC-associated microbiome research.

Frequency of MicroRNA Response Elements Identifies Pathologically Relevant Signaling Pathways in Triple-Negative Breast Cancer.

Complex interactions between mRNAs and microRNAs influence cellular functions. The mRNA-microRNA interactions also determine the post-transcriptional availability of mRNAs and unbound microRNAs. MicroRNAs binds to one or more microRNA response elements (MREs) located on the 3'UTR of mRNAs. In this study, we leveraged MREs and their frequencies in cancer and matched normal tissues to obtain insights into disease-specific interactions between mRNAs and microRNAs. We developed a bioinformatics method "ReMIx" that utilizes RNA sequencing (RNA-Seq) data to quantify MRE frequencies across the transcriptome. We applied ReMIx to triple-negative (TN) breast cancer tumor-normal adjacent pairs and identified MREs specific to TN tumors. ReMIx identified candidate mRNAs and microRNAs in the MAPK signaling cascade. Further analysis of MAPK gene regulatory networks revealed microRNA partners that influence and modulate MAPK signaling. In conclusion, we demonstrate a novel method of using MREs in the identification of functionally relevant mRNA-microRNA interactions in TN breast cancer.

Merit of an Ursodeoxycholic Acid Clinical Trial in COVID-19 Patients.

Corona Virus Disease 2019 (COVID-19) has affected over 8 million people worldwide. We underscore the potential benefits of conducting a randomized open-label unblinded clinical trial to evaluate the role of ursodeoxycholic acid (UDCA) in the treatment of COVID-19. Some COVID-19 patients are characterized with cytokine storm syndrome that can cause severe and irreversible damage to organs leading to multi-organ failure and death. Therefore, it is critical to control both programmed cell death (apoptosis) and the hyper-immune inflammatory response in COVID-19 patients to reduce the rising morbidity and mortality. UDCA is an existing drug with proven safety profiles that can reduce inflammation and prevent cell death. National Geographic reported that, "China Promotes Bear Bile as Coronavirus Treatment". Bear bile is rich in UDCA, comprising up to 40-50% of the total bile acid. UDCA is a logical and attainable replacement for bear bile that is available in pill form and merits clinical trial consideration.