Extracellular vesicles in colorectal cancer: immunomodulation, diagnostics, and therapeutic perspectives.

Editorial: Novel Clinical Applications of Extracellular Vesicles

Improving CAR-T Cell Function through a Targeted Cytokine Delivery System Utilizing CAR Target-Modified Extracellular Vesicles

Besides being powerhouses of the cell, mitochondria released into extracellular space act as intercellular signaling. Mitochondria and their components mediate cell-to-cell communication in free form or embedded in a carrier. The pathogenesis of cardiovascular disease is complex, which shows close relationship with inflammation and metabolic abnormalities. Since mitochondria sustain optimal function of the heart, extracellular mitochondria are emerging as a key regulator in the development of cardiovascular disease. In this review, we provide recent findings in the presence and forms of mitochondria transfer between cells, as well as the effects of these mitochondria on vascular inflammation and ischemic myocardium. Mitochondrial transplantation is a novel treatment paradigm for patients suffering from acute cardiovascular accident and challenges the traditional methods of mitochondria isolation.

Oncogenes can alter metabolism by changing the balance between anabolic and catabolic processes. However, how oncogenes regulate tumor cell biomass remains poorly understood. Using isogenic MCF10A cells transformed with nine different oncogenes, we show that specific oncogenes reduce the biomass of cancer cells by promoting extracellular vesicle (EV) release. While MYC and AURKB elicited the highest number of EVs, each oncogene selectively altered the protein composition of released EVs. Likewise, oncogenes alter secreted miRNAs. MYC-overexpressing cells require ceramide, whereas AURKB requires ESCRT to release high levels of EVs. We identify an inverse relationship between MYC upregulation and activation of the RAS/MEK/ERK signaling pathway for regulating EV release in some tumor cells. Finally, lysosome genes and activity are downregulated in the context of MYC and AURKB, suggesting that cellular contents, instead of being degraded, were released via EVs. Thus, oncogene-mediated biomass regulation via differential EV release is a new metabolic phenotype.

Targeted Delivery of IL-12 Via CD19-Modified Extracellular Vesicles Enhances CAR-T Cell Efficacy Against Lymphoma

Stimulated CAR-T cells from non-Hodgkin lymphomas patients engage and activate monocytes via extracellular vesicles

Circulating Extracellular Vesicles Induce Chimeric Antigen Receptor T Cell Dysfunction in Chronic Lymphocytic Leukemia (CLL)

Natural Killer-Cell Recovery in Patients Receiving CD19 CAR T-Cell Therapy: Dynamics and Clinical Significance

CD19.CAR T-cell–derived extracellular vesicles express CAR and kill leukemic cells, contributing to antineoplastic therapy

Despite the unprecedented clinical success of autologous chimeric antigen receptor (CAR) T cell therapy in B cell leukemia and lymphoma patients, CAR T cell therapy for acute myeloid leukemia (AML) patients has lagged, in part due to the time required for autologous CAR T cell preparation in the face of the rapid relapse of AML following remission, and the possibility of on-target off-tumor hematopoietic toxicity. In this study we investigated the functional activity of an allogeneic, off-the-shelf FLT3 CAR natural killer (NK) cells expressing soluble (s) IL-15 (FLT3 CAR_s15 NK) to treat FLT3+ AML. Cord blood NK cells were first transduced with FLT3 CAR intracellular signaling constructs containing either CD28/CD3ζ or 2B4/CD3ζ. Each displayed similar cytotoxicity against FLT3+ AML in vitro (p < 0.05) and in vivo (p < 0.01) when compared to mock transduced NK cells. Cord blood NK cells transduced with either soluble (s) IL-15 (s15 NK cells) or membrane (m) bound IL-15 showed comparable extended survival in vivo and superior to NK cells transduced with sIL-15/IL-15Rα (p < 0.01) or mIL-15/IL-15Rα (p < 0.01). However, only those NK cells transduced with soluble IL-15 were able to activate neighboring non-transduced (NT) NK cells and T cells in a paracrine fashion. The FLT3 CAR_s15 NK cells were expanded ex-vivo to over 1500-fold in 16 days with ~50% of the NK cells expressing both the FLT3 CAR and sIL-15. These FLT3 CAR_s15 NK cells were then subjected to a cytotoxicity assay against the FLT3+ AML cell line (MOLM-13) and were 10 times more potent than NT NK cells (p < 0.01) and 1.7 times more potent than s15 NK cells (p < 0.05). Likewise, when co-cultured with the FLT3+ AML cell line, FLT3 CAR_s15 NK cells produced a greater amount of IFN-γ compared to NT NK cells (p < 0.01) or compared to s15 NK cells (p < 0.05). Furthermore, following cryopreservation, our thawed FLT3 CAR_s15 NK cells demonstrated high recovery (~90%) and viability (> 85%), maintaining their FLT3 CAR expression at ~50% with a phenotype and cytotoxicity that was nearly equivalent to fresh FLT3 CAR_s15 NK cells. Armed with these data we assessed the efficacy of our allogeneic, off-the-shelf FLT3 CAR_s15 NK cells in vivo using immunodeficient mice engrafted with the MOLM-13 FLT3+ cell line. Repeated infusions of FLT3 CAR_s15 NK cells provided an improved median survival compared to placebo group (36 vs 24 days; p < 0.001) or compared to identical infusions of s15 NK cells (36 vs 31 days; p < 0.05) with similar results obtained in a patient-derived xenograft model. Finally, to assess toxicity against normal peripheral blood mononuclear cells (PBMC), including FLT3+ dendritic cells, and against hematopoietic stem cells (HSCs) we performed in vitro and in vivo studies, respectively, using our FLT3 CAR_s15 NK cells. We did not observe an untoward cytotoxicity of PBMC in vitro when compared to NT or s15 NK cells and no disruption of HSC differentiation in vivo when compared to placebo group. In summary, our in vitro and in vivo studies with allogeneic off-the-shelf cord blood derived FLT3 CAR_s15 NK cells demonstrated enhanced cytotoxicity and IFN-γ secretion against FLT3+ AML in vitro and enhanced the survival of mice engrafted with FLT3+ AML without any evidence of PBMC or HSC toxicity. We believe these data collectively support a rationale for investigating this novel form of allogeneic, off-the-shelf FLT3 CAR_s15 NK cell therapy in patients stricken with FLT3+ AML. Citation Format: Anthony G. Mansour, Kun-Yu Teng, Zhiyao Li, Zheng Zu, Hanyu Chen, Aliya Ali, Jianying Zhang, Ting Lu, Shoubao Ma, Michael A. Caligiuri, Jianhua Yu. Off-the-shelf cord blood FLT3 CAR-NK cells for immunotherapy of acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB102.

Introduction: Immune effector cell-associated neurotoxicity syndrome (ICANS) is a life-threatening adverse effect of anti-CD19 chimeric antigen receptor (CAR) T-cell therapy that usually occurs within 5–7 days after cell infusion. Although several clinical and biochemical parameters have been associated with ICANS, it is still a matter of debate how to predict its onset at the patient level. We here tested the hypothesis that CAR-T cell derived extracellular vesicles (EV) carrying the engineered CAR protein and produced early after CAR-T cell activation can be used as predictive biomarker of ICANS. Purposely, we measured plasma CAR+ EV in lymphoma patients underwent anti-CD19 CAR-T cell therapy. Methods: Seventy-one patients with aggressive r/r B-cell lymphomas were admitted to the advanced cell therapy unit of IRCCS AOU of Bologna (NCT04892433) for anti-CD19 CAR-T cell infusion. Included patients received tisa-cel (n = 27), axi-cel (n = 34), or brexu-cel (n = 10) after a median number of 3 prior lines of treatment (2–11); median age was 62 years (19–76) and no patients had CNS disease at the time of CAR-T cell infusion. Twenty out of 71 patients (28%) had ICANS of any grade: 5 patients (7%) ICANS grade 1, 7 patients (10%) ICANS grade 2 and 8 patients (11%) ICANS grade ≥3 (3 patients ICANS grade 3, 3 patients ICANS grade 4 and 2 patients ICANS grade 5 with diffuse cerebral edema). ICANS was classified according to Lee et al. The median time from CAR-T cell infusion to ICANS onset was 5 days (3–12). Available plasma samples at day +1 after CAR-T cell infusion were analyzed for CAR+ EV by FACS analysis. Data analysis was performed with Prism software v9.1.3 (GraphPad). Results: CAR+ EV were already detectable +1 day after CAR-T cell infusion in 58 patients. The median onset of ICANS was at day +5 (3–12). Patients with ICANS of any grade showed higher CAR+ EV level compared to no-ICANS ones (p < 0.0001). CAR+ EV anticipated the median ICANS onset of 2 to 11 days. CAR+ EV ROC analysis showed that a concentration >187.5 CAR+ EV/μl at day +1 after infusion predicts ICANS onset with sensitivity of 100% and specificity of 83.33% (p < 0.0001). Conclusions: These findings lead us to hypothesize that the plasma level of CAR+ EV mirrors target engagement by CAR-T cells, and their massive release is related to ICANS. Thus, CAR+ EV level could be considered a putative early predictor of ICANS onset; further analyses in larger cohorts are warranted to confirm this finding. Keywords: Cellular therapies, Diagnostic and Prognostic Biomarkers Conflicts of interests pertinent to the abstract. P. L. Zinzani Consultant or advisory role: Secura Bio, Celltrion, Gilead, Janssen-Cilag, BMS, Servier, Sandoz, MSD, AstraZeneca, Takeda, Roche, EUSA Pharma, Kyowa Kirin, Novartis, ADC Therapeutics, Incyte, BeiGene Other remuneration: Speakers bureau: Celltrion, Gilead, Janssen-Cilag, BMS, Servier, MSD, AstraZeneca, Takeda, Roche, EUSA Pharma, Kyowa Kirin, Novartis, Incyte, BeiGene F. Bonifazi Consultant or advisory role: Novartis, Gilead

Is there a true “shift” to the right colon in the incidence of colorectal cancer?

Previous studies have reported a migration in the occurrence of colorectal cancer (CRC) toward a proximal colonic location. To assess the potential impact of this, we evaluated recent temporal trends in the United States. Using the nine population-based cancer registries that constitute the Surveillance, Epidemiology, and End Results Program of the National Cancer Institute, we identified primary CRCs diagnosed between 1978 and 1998. Temporal changes were evaluated for 3-yr time periods. Age-adjusted incidence rates and the proportions of new diagnoses of CRC were calculated by site and by race. We identified 243,861 individuals with CRC during 1978-1998, 51.1% of whom were men. In whites and in blacks, the proportions of new diagnoses of right CRC rose significantly, from 34% and 37% to 40% and 44%, respectively. In contrast, the proportions of left CRC (in whites and blacks) and of rectosigmoid CRC (in whites) decreased significantly over time. For whites and blacks, the age-adjusted incidence rates for right CRC remained unchanged (in whites) or showed a small increase (in blacks), whereas the age-adjusted incidence rates for left CRC (in whites and blacks) and for rectosigmoid CRC (in whites) declined. During 1996-1998, right CRC was the most common site in the oldest age groups (70-79, 80 yr, and older) in whites and in blacks. The proximal migration of CRC over time is not attributed to a true increase in the incidence of right CRC. It is explained by a decrease in the incidence of distal CRC coupled with the aging of the population. Older individuals, in whom the burden of CRC is greatest, and in whom right CRC is the most common site, would be the most adversely affected from CRC screening methods that do not assess the total colon.

Colorectal cancer is the third most common cancer worldwide, with an estimated 1.36 million new cases and 694,000 deaths recorded in 2012. This number is expected to increase by 80% reaching approximately 2.4 million cases in 2035, and contributing to 1.3 million deaths globally per year. In Malaysia, colorectal cancer is the second most common cancer, in both men and women, behind lung cancer in men and breast cancer in women respectively. The most recent estimate gives the incidence of colorectal cancer in Malaysia as 21.3 cases per 100,000 population and 9.8 deaths per 100,000 population for the six years period reported between 2008 and 2013. The incidence of colorectal cancer is known to vary by place and by time period. However, most research on the spatial and variation in cancer incidence has taken place outside South East Asia, and such research on this is still scarce in Malaysia. The research presented in this thesis investigates individual factors affecting survival, and the spatial variation in incidence and survival of colorectal cancer in Malaysia. This has not been done before in Malaysia. There are three objectives addressed in this thesis: (1) to investigate the individual characteristics that affect survival of colorectal cancer in Malaysia, (2) to investigate and model the spatial variation of survival in colorectal cancer in Malaysia, (3) to investigate and model the spatial variation in the incidence of colorectal cancer in Malaysia. The research was involved 4412 of colorectal cancer patients in Malaysia with histologically verified primary colorectal cancer who were diagnosed between 2008 and 2013 (ICD-10, C18-C20), recorded in the database of National Cancer Patient Registry- Colorectal Cancer (NCPR-CC) Malaysia. We investigated the effect of individual characteristics such as age, gender, education as well as clinical characteristics such as cancer staging, cancer site and treatment modalities on survival prognosis after a diagnosis of colorectal cancer using a Cox regression model. The analysis was then been extended to model the spatial variation in survival for colorectal cancer patients in Malaysia, accounting for individual and socioeconomic characteristics using a spatial survival model. We then applied a Generalized Linear Mixed Effects model, which is derived from the log-Gaussian Cox Process, in order to model the incidence of colorectal cancer in Peninsular Malaysia. Our findings show that the severity of disease at diagnosis as measured by cancer staging, tumour grading and the presence of distant metastases, plays an important role in the prognosis of patients with colorectal cancer in Malaysia, and that this remains even after controlling for spatial correlation on space. Our research allows us to shows the geographical variation in survival of colorectal cancer in Malaysia, and what variation persists once individual and socioeconomic characteristics are taken into account. Our model that developed to predict the colorectal cancer incidence found that that some places had greater risk of an incidence exceeding the national average. The map for probability of exceedance relative risk of colorectal cancer incidence in the North West of Peninsular Malaysia shows variation in the risk for colorectal cancer cases across the region. We noted that town areas are highly likely to exceed the threshold of relative risk of increased number of colorectal cancer cases, and this effect is present even though we account for the additional population there. Spatial variation in survival and incidence of colorectal cancer in Malaysia needs to be investigated further. To the best of our knowledge, this is the first research that uses spatial modelling to identify potential factors affecting the incidence and survival for colorectal cancer in Malaysia, as well as to map the risks in survival and incidence. Our findings can help public health authorities to plan better management of the resources used to prevent and treat this disease.

Extracellular vesicles (EVs) are nano-sized membrane particles secreted by various cell types that are involved in many important cellular processes. Recently, EVs originating from immune cells, such as dendritic cells, chimeric antigen receptor T cells, and natural killer cells, have attracted much attention because of their known direct and indirect antitumor activity. Here, we report the EVs released by cytokine-activated CD8+ T (caCD8) cells and its cytotoxicity against cancer cells. CaCD8 cells can release EVs following stimulation of CD8+ T cells with an anti-CD3 antibody and a cytokine cocktail ex vivo. The isolated vesicles have typical EV characteristics, such as an oval shape and a size distribution between 30 and 200 nm, as well as CD81 expression. Notably, caCD8-EVs displayed cytotoxicity against various cancer cells in vitro. Furthermore, mechanism analysis demonstrates that caCD8-EVs not only contain typical cytotoxic proteins (i.e. granzyme B and perforin), but also significantly enrich interferon γ (IFNγ) compared with caCD8 cells. EV-derived IFNγ participates in EV-induced apoptosis in cancer cells. Therefore, our data reveal antitumor effects of EVs secreted from caCD8 cells and the potential role of the EV-derived IFNγ.