Engineered immunomodulatory extracellular vesicles derived from epithelial cells acquire capacity for positive and negative T cell co-stimulation in cancer and autoimmunity.

Extracellular vesicles (EVs) are generated by all cells and systemic administration of allogenic EVs derived from epithelial and mesenchymal cells have been shown to be safe, despite carrying an array of functional molecules, including thousands of proteins. To address whether epithelial cells derived EVs can be modified to acquire the capacity to induce immune response, we engineered 293T EVs to harbor the immunomodulatory CD80, OX40L and PD-L1 molecules. We demonstrated abundant levels of these proteins on the engineered cells and EVs. Functionally, the engineered EVs efficiently elicit positive and negative co-stimulation in human and murine T cells. In the setting of cancer and auto-immune hepatitis, the engineered EVs modulate T cell functions and alter disease progression. Moreover, OX40L EVs provide additional benefit to anti-CTLA-4 treatment in melanoma-bearing mice. Our work provides evidence that epithelial cell derived EVs can be engineered to induce immune responses with translational potential to modulate T cell functions in distinct pathological settings.

KRASG12D inhibition reprograms the microenvironment of early and advanced pancreatic cancer to promote FAS-mediated killing by CD8+ T cells.

The KRASG12D mutation is present in nearly half of pancreatic adenocarcinomas (PDAC). We investigated the effects of inhibiting the KRASG12D mutant protein with MRTX1133, a non-covalent small molecule inhibitor of KRASG12D, on early and advanced PDAC and its influence on the tumor microenvironment. Employing 16 different models of KRASG12D-driven PDAC, we demonstrate that MRTX1133 reverses early PDAC growth, increases intratumoral CD8+ effector T cells, decreases myeloid infiltration, and reprograms cancer-associated fibroblasts. MRTX1133 leads to regression of both established PanINs and advanced PDAC. Regression of advanced PDAC requires CD8+ T cells and immune checkpoint blockade (ICB) synergizes with MRTX1133 to eradicate PDAC and prolong overall survival. Mechanistically, inhibition of KRASG12D in advanced PDAC and human patient derived organoids induces FAS expression in cancer cells and facilitates CD8+ T cell-mediated death. Collectively, this study provides a rationale for a synergistic combination of MRTX1133 with ICB in clinical trials.

Elimination of oncogenic KRAS in genetic mouse models eradicates pancreatic cancer by inducing FAS-dependent apoptosis by CD8+ T cells.

Oncogenic KRASG12D (KRAS∗) is critical for the initiation and maintenance of pancreatic ductal adenocarcinoma (PDAC) and is a known repressor of tumor immunity. Conditional elimination of KRAS∗ in genetic mouse models of PDAC leads to the reactivation of FAS, CD8+ T cell-mediated apoptosis, and complete eradication of tumors. KRAS∗ elimination recruits activated CD4+ and CD8+ T cells and promotes the activation of antigen-presenting cells. Mechanistically, KRAS∗-mediated immune evasion involves the epigenetic regulation of Fas death receptor in cancer cells, via methylation of its promoter region. Furthermore, analysis of human RNA sequencing identifies that high KRAS expression in PDAC tumors shows a lower proportion of CD8+ T cells and demonstrates shorter survival compared with tumors with low KRAS expression. This study highlights the role of CD8+ T cells in the eradication of PDAC following KRAS∗ elimination and provides a rationale for the combination of KRAS∗ targeting with immunotherapy to control PDAC.

Identification of unique α4 chain structure and conserved antiangiogenic activity of α3NC1 type IV collagen in zebrafish.

BACKGROUND: Type IV collagen is an abundant component of basement membranes in all multicellular species and is essential for the extracellular scaffold supporting tissue architecture and function. Lower organisms typically have two type IV collagen genes, encoding α1 and α2 chains, in contrast with the six genes in humans, encoding α1-α6 chains. The α chains assemble into trimeric protomers, the building blocks of the type IV collagen network. The detailed evolutionary conservation of type IV collagen network remains to be studied. RESULTS: We report on the molecular evolution of type IV collagen genes. The zebrafish α4 non-collagenous (NC1) domain, in contrast with its human ortholog, contains an additional cysteine residue and lacks the M93 and K211 residues involved in sulfilimine bond formation between adjacent protomers. This may alter α4 chain interactions with other α chains, as supported by temporal and anatomic expression patterns of collagen IV chains during the zebrafish development. Despite the divergence between zebrafish and human α3 NC1 domain (endogenous angiogenesis inhibitor, Tumstatin), the zebrafish α3 NC1 domain exhibits conserved antiangiogenic activity in human endothelial cells. CONCLUSIONS: Our work supports type IV collagen is largely conserved between zebrafish and humans, with a possible difference involving the α4 chain.

Oncogenic Kras G12D specific non-covalent inhibitor reprograms tumor microenvironment to prevent and reverse early pre-neoplastic pancreatic lesions and in combination with immunotherapy regresses advanced PDAC in a CD8 + T cells dependent manner.

Pancreatic ductal adenocarcinoma (PDAC) is associated with mutations in Kras, a known oncogenic driver of PDAC; and the KRAS G12D mutation is present in nearly half of PDAC patients. Recently, a non-covalent small molecule inhibitor (MRTX1133) was identified with specificity to the Kras G12D mutant protein. Here we explore the impact of Kras G12D inhibition by MRTX1133 on advanced PDAC and its influence on the tumor microenvironment. Employing different orthotopic xenograft and syngeneic tumor models, eight different PDXs, and two different autochthonous genetic models, we demonstrate that MRTX1133 reverses early PDAC growth, increases intratumoral CD8 + effector T cells, decreases myeloid infiltration, and reprograms cancer associated fibroblasts. Autochthonous genetic mouse models treated with MRTX1133 leads to regression of both established PanINs and advanced PDAC. Regression of advanced PDAC requires CD8 + T cells and immune checkpoint blockade therapy (iCBT) synergizes with MRTX1133 to eradicate PDAC and prolong overall survival. Mechanistically, inhibition of mutant Kras in advanced PDAC and human patient derived organoids (PDOs) induces Fas expression in cancer cells and facilitates CD8 + T cell mediated death. These results demonstrate the efficacy of MRTX1133 in different mouse models of PDAC associated with reprogramming of stromal fibroblasts and a dependency on CD8 + T cell mediated tumor clearance. Collectively, this study provides a rationale for a synergistic combination of MRTX1133 with iCBT in clinical trials.

Plasticity Enables Cooperation among Heterogeneous Cancer Cell Populations to Support Metastatic Fitness.

The invasive progression of cancer known as metastasis remains strongly associated with morbidity and lethality. New meaningful therapeutic interventions could be derived from a better understanding of the underlying processes driving cancer cell seeding and proliferation at secondary sites. Emerging findings regarding the heterogeneity of cancer cells observed in metastases have led us to revisit concepts surrounding metastatic fitness. Novel model systems to study the markers of cancer stem cell plasticity and their evolution during metastatic growth have uncovered that dynamic and heterogeneous cancer cell populations are observed during metastatic disease progression. Heinz and colleagues studied the heterogeneity of colorectal carcinomas, where primary tumors evolve alongside an epithelium well characterized for its self-renewing stem cell population. Their work revealed a functional dynamic interplay in the organization of the metastatic lesions as they transition from stagnating to expanding nodules, wherein the heterogeneous mixture of cancer cell stem cells with more differentiated cancer cells is essential for metastatic outgrowth. Their work supports that dynamic YAP signaling enables the growth-permissive heterogeneous composition of the metastatic nodule, in contrast with growth-restricted homogeneous compositions. See related article by Heinz et al., p. 1953.

The Continuing Search for Causality between Epithelial-to-Mesenchymal Transition and the Metastatic Fitness of Carcinoma Cells.

The epithelial-to-mesenchymal transition (EMT) is an epithelial plasticity program that is associated with embryonic development and organogenesis, and which resurfaces to a certain extent following epithelial injury caused by inflammation, fibrosis, and carcinoma progression. Carcinoma cell EMT plasticity programs superimposed on inherent genetic defects have been implicated as important for metastatic dissemination and secondary tumor formation. A careful review of data-driven facts suggests that a causal relationship between any degree of EMT program and metastasis continues to be elusive, and the steps of metastasis likely involve other mechanisms influenced by the carcinoma cell genotype and the tumor microenvironment.

Identification of Functional Heterogeneity of Carcinoma-Associated Fibroblasts with Distinct IL6-Mediated Therapy Resistance in Pancreatic Cancer.

The tumor microenvironment in pancreatic ductal adenocarcinoma (PDAC) involves a significant accumulation of fibroblasts as part of the host response to cancer. Using single-cell RNA sequencing, multiplex immunostaining, and several genetic mouse models, we identify carcinoma-associated fibroblasts (CAF) with opposing functions in PDAC progression. Depletion of fibroblast activation protein (FAP)+ CAFs results in increased survival, in contrast to depletion of alpha smooth muscle actin (αSMA)+ CAFs, which leads to decreased survival. Tumor-promoting FAP+ CAFs (TP-CAF) and tumor-restraining αSMA+ CAFs (TR-CAF) differentially regulate cancer-associated pathways and accumulation of regulatory T cells. Improved efficacy of gemcitabine is observed when IL6 is deleted from αSMA+ CAFs but not from FAP+ CAFs using dual-recombinase genetic PDAC models. Improved gemcitabine efficacy due to lack of IL6 synergizes with anti-PD-1 immunotherapy to significantly improve survival of PDAC mice. Our study identifies functional heterogeneity of CAFs in PDAC progression and their different roles in therapy response. SIGNIFICANCE: PDAC is associated with accumulation of dense stroma consisting of fibroblasts and extracellular matrix that regulate tumor progression. Here, we identify two distinct populations of fibroblasts with opposing roles in the progression and immune landscape of PDAC. Our findings demonstrate that fibroblasts are functionally diverse with therapeutic implications. This article is highlighted in the In This Issue feature, p. 1397.

Engineered exosomes targeting MYC reverse the proneural-mesenchymal transition and extend survival of glioblastoma.

Dysregulated Myc signaling is a key oncogenic pathway in glioblastoma multiforme (GBM). Yet, effective therapeutic targeting of Myc continues to be challenging. Here, we demonstrate that exosomes generated from human bone marrow mesenchymal stem cells (MSCs) engineered to encapsulate siRNAs targeting Myc (iExo-Myc) localize to orthotopic GBM tumors in mice. Treatment of late stage GBM tumors with iExo-Myc inhibits proliferation and angiogenesis, suppresses tumor growth, and extends survival. Transcriptional profiling of tumors reveals that the mesenchymal transition and estrogen receptor signaling pathways are impacted by Myc inhibition. Single nuclei RNA sequencing (snRNA-seq) shows that iExo-Myc treatment induces transcriptional repression of multiple growth factor and interleukin signaling pathways, triggering a mesenchymal to proneural transition and shifting the cellular landscape of the tumor. These data confirm that Myc is an effective anti-glioma target and that iExo-Myc offers a feasible, readily translational strategy to inhibit challenging oncogene targets for the treatment of brain tumors.

Unique somatic variants in DNA from urine exosomes of individuals with bladder cancer.

Bladder cancer (BC), a heterogeneous disease characterized by high recurrence rates, is diagnosed and monitored by cystoscopy. Accurate clinical staging based on biopsy remains a challenge, and additional, objective diagnostic tools are needed urgently. We used exosomal DNA (exoDNA) as an analyte to examine cancer-associated mutations and compared the diagnostic utility of exoDNA from urine and serum of individuals with BC. In contrast to urine exosomes from healthy individuals, urine exosomes from individuals with BC contained significant amounts of DNA. Whole-exome sequencing of DNA from matched urine and serum exosomes, bladder tumors, and normal tissue (peripheral blood mononuclear cells) identified exonic and 3' UTR variants in frequently mutated genes in BC, detectable in urine exoDNA and matched tumor samples. Further analyses identified somatic variants in driver genes, unique to urine exoDNA, possibly because of the inherent intra-tumoral heterogeneity of BC, which is not fully represented in random small biopsies. Multiple variants were also found in untranslated portions of the genome, such as microRNA (miRNA)-binding regions of the KRAS gene. Gene network analyses revealed that exoDNA is associated with cancer, inflammation, and immunity in BC exosomes. Our findings show utility of exoDNA as an objective, non-invasive strategy to identify novel biomarkers and targets for BC.

Exosome-mediated delivery of CRISPR/Cas9 for targeting of oncogenic KrasG12D in pancreatic cancer.

CRISPR/Cas9 is a promising technology for gene editing. To date, intracellular delivery vehicles for CRISPR/Cas9 are limited by issues of immunogenicity, restricted packaging capacity, and low tolerance. Here, we report an alternative, nonviral delivery system for CRISPR/Cas9 based on engineered exosomes. We show that non-autologous exosomes can encapsulate CRISPR/Cas9 plasmid DNA via commonly available transfection reagents and can be delivered to recipient cancer cells to induce targeted gene deletion. As a proof-of-principle, we demonstrate that exosomes loaded with CRISPR/Cas9 can target the mutant Kras G12D oncogenic allele in pancreatic cancer cells to suppress proliferation and inhibit tumor growth in syngeneic subcutaneous and orthotopic models of pancreatic cancer. Exosomes may thus be a promising delivery platform for CRISPR/Cas9 gene editing for targeted therapies.

αSMA+ fibroblasts suppress Lgr5+ cancer stem cells and restrain colorectal cancer progression.

The development and progression of solid tumors is dependent on cancer cell autonomous drivers and the tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs) in the TME possess both tumor-promoting and tumor-restraining functions. In the current study, we interrogated the role of αSMA+ CAFs in a genetic mouse model of metastatic colorectal cancer (CRC). Selective depletion of αSMA+ CAFs resulted in increased tumor invasiveness, lymph node metastasis, and reduced overall survival. Depletion of αSMA+ CAFs reduced BMP4 and increased TGFß1 secretion from stromal cells, and was associated with increased Lgr5+ cancer stem-like cells (CSCs) and the generation of an immunosuppressive TME with increased frequency of Foxp3+ regulatory T cells and suppression of CD8+ T cells. This study demonstrates that αSMA+ CAFs in CRC exert tumor-restraining functions via BMP4/TGFß1 paracrine signaling that serves to suppress Lgr5+ CSCs and promote anti-tumor immunity, ultimately limiting CRC progression.

Type I collagen deletion in αSMA+ myofibroblasts augments immune suppression and accelerates progression of pancreatic cancer.

Stromal desmoplastic reaction in pancreatic ductal adenocarcinoma (PDAC) involves significant accumulation of type I collagen (Col1). However, the precise molecular and mechanistic contribution of Col1 in PDAC progression remains unknown. Activated pancreatic stellate cells/αSMA+ myofibroblasts are major contributors of Col1 in the PDAC stroma. We use a dual-recombinase genetic mouse model of spontaneous PDAC to delete Col1 specifically in myofibroblasts. This results in significant reduction of total stromal Col1 content and accelerates the emergence of PanINs and PDAC, decreasing overall survival. Col1 deletion leads to Cxcl5 upregulation in cancer cells via SOX9. Increase in Cxcl5 is associated with recruitment of myeloid-derived suppressor cells and suppression of CD8+ T cells, which can be attenuated with combined targeting of CXCR2 and CCR2 to restrain accelerated PDAC progression in the setting of stromal Col1 deletion. Our results unravel the fundamental role of myofibroblast-derived Co1l in regulating tumor immunity and restraining PDAC progression.

Effective delivery of STING agonist using exosomes suppresses tumor growth and enhances antitumor immunity.

The Stimulator of Interferon Genes (STING) pathway is implicated in the innate immune response and is important in both oncogenesis and cancer treatment. Specifically, activation of the cytosolic DNA sensor STING in antigen-presenting cells (APCs) induces a type I interferon response and cytokine production that facilitates antitumor immune therapy. However, use of STING agonists (STINGa) as a cancer therapeutic has been limited by unfavorable pharmacological properties and targeting inefficiency due to rapid clearance and limited uptake into the cytosol. Exosomes, a class of extracellular vesicles shed by all cells are under consideration for their use as effective carriers of drugs owing to their innate ability to be taken up by cells and their biocompatibility for optimal drug biodistribution. Therefore, we engineered exosomes to deliver the STING agonist cyclic GMP-AMP (iExoSTINGa), to exploit their favorable pharmacokinetics and pharmacodynamics. Selective targeting of the STING pathway in APCs with iExoSTINGa was associated with superior potency compared with STINGa alone in suppressing B16F10 tumor growth. Moreover, iExoSTINGa showed superior uptake of STINGa into dendritic cells compared with STINGa alone, which led to increased accumulation of activated CD8+ T-cells and an antitumor immune response. Our study highlights the potential of exosomes in general, and iExoSTINGa specifically, in enhancing cancer therapy outcomes.

Protection against SARS-CoV-2 by BCG vaccination is not supported by epidemiological analyses.

The Bacillus Calmette-Guerin (BCG) vaccine provides protection against tuberculosis (TB), and is thought to provide protection against non-TB infectious diseases. BCG vaccination has recently been proposed as a strategy to prevent infection with SARS-CoV-2 (CoV-2) to combat the COVID-19 outbreak, supported by its potential to boost innate immunity and initial epidemiological analyses which observed reduced severity of COVID-19 in countries with universal BCG vaccination policies. Seventeen clinical trials are currently registered to inform on the benefits of BCG vaccinations upon exposure to CoV-2. Numerous epidemiological analyses showed a correlation between incidence of COVID-19 and BCG vaccination policies. These studies were not systematically corrected for confounding variables. We observed that after correction for confounding variables, most notably testing rates, there was no association between BCG vaccination policy and COVD-19 spread rate or percent mortality. Moreover, we found variables describing co-morbidities, including cardiovascular death rate and smoking prevalence, were significantly associated COVID-19 spread rate and percent mortality, respectively. While reporting biases may confound our observations, our epidemiological findings do not provide evidence to correlate overall BCG vaccination policy with the spread of CoV-2 and its associated mortality.

Endothelial-to-mesenchymal transition compromises vascular integrity to induce Myc-mediated metabolic reprogramming in kidney fibrosis.

Endothelial-to-mesenchymal transition (EndMT) is a cellular transdifferentiation program in which endothelial cells partially lose their endothelial identity and acquire mesenchymal-like features. Renal capillary endothelial cells can undergo EndMT in association with persistent damage of the renal parenchyma. The functional consequence(s) of EndMT in kidney fibrosis remains unexplored. Here, we studied the effect of Twist or Snail deficiency in endothelial cells on EndMT in kidney fibrosis. Conditional deletion of Twist1 (which encodes Twist) or Snai1 (which encodes Snail) in VE-cadherin+ or Tie1+ endothelial cells inhibited the emergence of EndMT and improved kidney fibrosis in two different kidney injury/fibrosis mouse models. Suppression of EndMT limited peritubular vascular leakage, reduced tissue hypoxia, and preserved tubular epithelial health and function. Hypoxia, which was exacerbated by EndMT, resulted in increased Myc abundance in tubular epithelial cells, enhanced glycolysis, and suppression of fatty acid oxidation. Pharmacological suppression or epithelial-specific genetic ablation of Myc in tubular epithelial cells ameliorated fibrosis and restored renal parenchymal function and metabolic homeostasis. Together, these findings demonstrate a functional role for EndMT in the response to kidney capillary endothelial injury and highlight the contribution of endothelial-epithelial cross-talk in the development of kidney fibrosis with a potential for therapeutic intervention.

Epigenetic Reprogramming of Cancer-Associated Fibroblasts Deregulates Glucose Metabolism and Facilitates Progression of Breast Cancer.

The mechanistic contributions of cancer-associated fibroblasts (CAFs) in breast cancer progression remain to be fully understood. While altered glucose metabolism in CAFs could fuel cancer cells, how such metabolic reprogramming emerges and is sustained needs further investigation. Studying fibroblasts isolated from patients with benign breast tissues and breast cancer, in conjunction with multiple animal models, we demonstrate that CAFs exhibit a metabolic shift toward lactate and pyruvate production and fuel biosynthetic pathways of cancer cells. The depletion or suppression of the lactate production of CAFs alter the tumor metabolic profile and impede tumor growth. The glycolytic phenotype of the CAFs is in part sustained through epigenetic reprogramming of HIF-1α and glycolytic enzymes. Hypoxia induces epigenetic reprogramming of normal fibroblasts, resulting in a pro-glycolytic, CAF-like transcriptome. Our findings suggest that the glucose metabolism of CAFs evolves during tumor progression, and their breast cancer-promoting phenotype is partly mediated by oxygen-dependent epigenetic modifications.

Exosomes as a Multicomponent Biomarker Platform in Cancer.

Cancer is a complex disease that is associated with genetic aberrations and subsequent cellular and noncellular host responses. Tumors harbor diverse cell types that engage in a dynamic interplay to sustain cancer-specific signaling networks. A component of such cellular communication is the production and exchange of various types of extracellular vesicle (EV). Exosomes are small EVs with growing recognition for their role in cancer progression and resistance to therapy. The unique biogenesis of exosomes, their ubiquitous production by all cell types, and their biological features in liquid biopsies have generated excitement for their potential as cancer biomarkers. Here, we discuss the challenges and utility of exosomes as multiparameter biomarker platforms for the detection of cancer. Exosomes reflect heterogeneous biological changes associated with growing tumors, potentially offering a more comprehensive assessment of cancer diagnosis, prognosis, and progression.