Antigen-presenting cells phagocytose tumor cells and subsequently cross-present tumor-derived antigens. However, these processes are impeded by phagocytosis checkpoints and inefficient cytosolic transport of antigenic peptides from phagolysosomes. Here, using a microbial-inspired strategy, we engineered an antibody–toxin conjugate (ATC) that targets the ‘don’t eat me’ signal CD47 linked to the bacterial toxin listeriolysin O from the intracellular bacterium Listeria monocytogenes via a cleavable linker (CD47–LLO). CD47–LLO promotes cancer cell phagocytosis by macrophages followed by LLO release and activation to form pores on phagolysosomal membranes that enhance antigen cross-presentation of tumor-derived peptides and activate cytosolic immune sensors. CD47–LLO treatment in vivo significantly inhibited the growth of both localized and metastatic breast and melanoma tumors and improved animal survival as a monotherapy or in combination with checkpoint blockade. Together, these results demonstrate that designing ATCs to promote immune recognition of tumor cells represents a promising therapeutic strategy for treating multiple cancers. Schrank et al. report the design and characterization of an antibody–toxin conjugate targeting CD47, promoting anti-tumor immunity in preclinical cancer models.

Intracellular DNA sensors regulate innate immunity and can provide a bridge to adaptive immunogenicity. However, the activation of the sensors in antigen-presenting cells (APCs) by natural agonists such as double-stranded DNAs or cyclic nucleotides is impeded by poor intracellular delivery, serum stability, enzymatic degradation and rapid systemic clearance. Here we show that the hydrophobicity, electrostatic charge and secondary conformation of helical polypeptides can be optimized to stimulate innate immune pathways via endoplasmic reticulum stress in APCs. One of the three polypeptides that we engineered activated two major intracellular DNA-sensing pathways (cGAS-STING (for cyclic guanosine monophosphate-adenosine monophosphate synthase-stimulator of interferon genes) and Toll-like receptor 9) preferentially in APCs by promoting the release of mitochondrial DNA, which led to the efficient priming of effector T cells. In syngeneic mouse models of locally advanced and metastatic breast cancers, the polypeptides led to potent DNA-sensor-mediated antitumour responses when intravenously given as monotherapy or with immune checkpoint inhibitors. The activation of multiple innate immune pathways via engineered cationic polypeptides may offer therapeutic advantages in the generation of antitumour immune responses.

Supplementary Table 7: DEG in LDE vs. control treated SI-CSC Ptch;p53 tumors

Supplementary Table 8: DEG in LDE vs control in SD-CSC Ptch;p53 tumors

Nanomedicines have been approved to treat multiple human diseases. However, clinical adoption of nanoformulated agents is often hindered by concerns about hepatic uptake and clearance, a process that is not fully understood. Here we show that the antitumour efficacy of cancer nanomedicine exhibits an age-associated disparity. Tumour delivery and treatment outcomes are superior in old versus young mice, probably due to an age-related decline in the ability of hepatic phagocytes to take up and remove nanoparticles. Transcriptomic- and protein-level analysis at the single-cell and bulk levels reveals an age-associated decrease in the numbers of hepatic macrophages that express the scavenger receptor MARCO in mice, non-human primates and humans. Therapeutic blockade of MARCO is shown to decrease the phagocytic uptake of nanoparticles and improve the antitumour effect of clinically approved cancer nanotherapeutics in young but not aged mice. Together, these results reveal an age-associated disparity in the phagocytic clearance of nanotherapeutics that affects their antitumour response, thus providing a strong rationale for an age-appropriate approach to cancer nanomedicine.

Immunotherapies have provided dramatic and life-saving results in some patients; however, their effectiveness varies greatly in different tumors, even among patients with the same clinical diagnosis. Clearly, a better system to evaluate the personalized response to immunotherapies will greatly benefit the patients and reduce the cost of care. In addition, due to their theoretical and demonstrated benefits in providing durable responses, new immunotherapies are avidly sought after by most major pharmaceutical companies, numerous biotech companies, and academic laboratories. However, preclinical evaluation of new immunotherapy efficacy is challenging and inaccurate using most experimental model systems.E-slice is a faithful model of an individual cancer patient’s tumor. It is a proprietary 3D tissue slice culture platform, and E-slices are generated by making thin sections of intact, fresh tumor tissues from patients. As such, immune components and the TME in E-slices are native to each patient’s tumor. E-slice overcomes many of the limitations of other experimental systems for multiple reasons. The E-slice platform: 1) uses chemically defined, serum-free medium; 2) measures viability changes upon treatment longitudinally, from the same tissue, which allows both absolute and relative responses for as short as 4 days or as long as over 4 weeks ex vivo; 3) can be generated from any solid tumor tested thus far (breast, lung, colorectal, pancreas, brain, head 7 neck, and others) from patient tumors directly or from PDX and genetically engineered mouse models; 4) retains the native TME and tissue architecture because E-slices are never dissociated or otherwise reconstituted; 5) is compatible with biopsies as well as surgical samples; 6) has been shown to accurately predict individual patient treatment responses to chemotherapies and targeted therapies in 4-12 days, paving the way for evidence-based personalized treatment selections in a clinically actionable time frame.Specifically for immunotherapy response measurements, key differentiators of the E-slice platform from other ex vivo systems for immunotherapy responses are: 1) E-slice is generated from fresh patient tumor tissues with tumor-trained and resident immune cells and not artificially introduced PBMCs used in most other systems; 2) serum-free, chemically defined medium and does not artificially activate or suppress the immune system; 3) E-slice has been validated by single-cell RNA-sequencing to maintain immune cells in their native state up to 8 days ex vivo; 4) E-slices can be used to detect secreted protein and metabolic biomarkers in the conditioned media pre- and post-treatment; 5) E-slice can measure immunotherapy responses ex vivo in 8 days. In summary, we present a novel ex vivo 3D human tumor tissue drug sensitivity platform that can enhance immunotherapy development pipelines and clinical deployment. Citation Format: Thomas D. Gallup, Jose A. Maldonado, Corina Margain, Min P. Kim, David F. Gallup, Kyuson Yun. Measuring immunotherapy responses ex vivo using novel 3D culture platform: E-slice. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4563.

Supplementary Table 5: mutational profiles for Fsmo;GFAP-cre SD-CSC tumors

Supplementary Table 4: mutational profiles for Ptch;p53 SI-CSC tumors

Supplementary Table 3: Mutational profiles for Ptch;p53 SD-CSC tumors

Supplementary Table 9: Differentially expressed proteins in LDE vs. control treated Ptch;p53 SI-CSC and SD-CSC tumors

The success of messenger RNA therapeutics largely depends on the availability of delivery systems that enable the safe, effective and stable translation of genetic material into functional proteins. Here we show that extracellular vesicles (EVs) produced via cellular nanoporation from human dermal fibroblasts, and encapsulating mRNA encoding for extracellular-matrix α1 type-I collagen (COL1A1) induced the formation of collagen-protein grafts and reduced wrinkle formation in the collagen-depleted dermal tissue of mice with photoaged skin. We also show that the intradermal delivery of the mRNA-loaded EVs via a microneedle array led to the prolonged and more uniform synthesis and replacement of collagen in the dermis of the animals. The intradermal delivery of EV-based COL1A1 mRNA may make for an effective protein-replacement therapy for the treatment of photoaged skin.

Supplementary figures and Tables 1 & 2

Supplementary Table 6: DEG in LDE vs control treated fSMO;GFAP-cre tumors

The need for personalized medicine in oncology is widely accepted but translating this important concept into clinical practice has been challenging. Currently, the dominant platform for precision medicine utilizes genomics/sequencing-based assays to measure the expression and/or mutational profiles and then infer patient responses to therapies based on previous knowledge; however, this approach benefits less than 10-15% of patients with profiled tumors. Recognizing the inherent limitations of these inference-based methods, functional assays (e.g., organoids and PDX models) have been developed; however, these approaches also have significant limitations including high cost and time required to establish the models, low “take rates”, and destruction of the native tumor microenvironment (TME). To overcome these challenges, EMPIRI uses a novel 3D ex vivo tumor slice culture method (E-slices) that enables rapid, personalized drug sensitivity testing in intact patient tumor tissues. Major differentiators of the E-slice platform from other ex vivo methods include the use of chemically defined, serum-free medium, longitudinal viability measurements from the same tissue, tracking of dynamic responses to treatment over 2-3 weeks, and retention of the native TME and tissue architecture, unlike other approaches. In addition, E-slices can be generated from any solid tumor tested thus far (breast, lung, colorectal, pancreas, brain, head 7 neck, and others) from patient tumors directly and PDX and genetically engineered mouse models. In addition, because E-slices retain tumor-infiltrating immune cells in their native microenvironment and spatial topography of all cell types in the endogenous configuration, it sustains immune cell survival and proliferation and measures immunotherapy responses ex vivo. The E-slice method is compatible with biopsies as well as surgical samples. Importantly, it has been shown to accurately predict individual patient treatment responses to chemotherapies and targeted therapies in 4-12 days, paving the way for evidence-based personalized treatment selections in a clinically actionable time frame. In summary, we present a novel ex vivo 3D human tumor tissue drug sensitivity platform that faithfully replicates the patient tumor tissues and provides personalized treatment responses in a clinically actionable time frame. Citation Format: Archana Gopalan, Thomas D. Gallup, Stephanie Wood, Jose Maldonado, Corina Margain, Nestor F. Esnaola, Min P. Kim, E. Scott Kopetz, Kyuson Yun. E-slice: A novel 3D culture platform for precision medicine [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 694.

The emergence of treatment resistance significantly reduces the clinical utility of many effective targeted therapies. Although both genetic and epigenetic mechanisms of drug resistance have been reported, whether these mechanisms are stochastically selected in individual tumors or governed by a predictable underlying principle is unknown. Here, we report that the dependence of cancer stem cells (CSC), not bulk tumor cells, on the targeted pathway determines the molecular mechanism of resistance in individual tumors. Using both spontaneous and transplantable mouse models of sonic hedgehog (SHH) medulloblastoma treated with a SHH/Smoothened inhibitor (SMOi), sonidegib/LDE225, we show that genetic-based resistance occurs only in tumors that contain SHH-dependent CSCs. In contrast, SHH medulloblastomas containing SHH-dependent bulk tumor cells but SHH-independent CSCs (SI-CSC) acquire resistance through epigenetic reprogramming. Mechanistically, elevated proteasome activity in SMOi-resistant SI-CSC medulloblastomas alters the tumor cell maturation trajectory through enhanced degradation of specific epigenetic regulators, including histone acetylation machinery components, resulting in global reductions in H3K9Ac, H3K14Ac, H3K56Ac, H4K5Ac, and H4K8Ac marks and gene expression changes. These results provide new insights into how selective pressure on distinct tumor cell populations contributes to different mechanisms of resistance to targeted therapies. This insight provides a new conceptual framework to understand responses and resistance to SMOis and other targeted therapies. Significance: The mechanism by which individual tumors become resistant to targeted therapies is thought to be unpredictable. This study provides novel insights into how selective pressure on cancer stem versus bulk tumor cells drives distinct and predictable mechanisms of resistance to targeted therapies. This finding paves a way for future treatment strategies that incorporate anticipated resistance mechanisms in devising second-line therapies in a personalized manner.

Immunotherapy is a promising treatment modality for highly invasive gliomas; however, clinical trials thus far have failed to provide significant clinical benefit to most GBM patients. GBM is one of the “immune cold” tumors characterized by poor infiltration of T cells even though greater than 40% of glioma cells are composed of immune cells in some patients. The majority of immune cells in GBM are immune-suppressive myeloid cells that block T cell infiltration and/or activation. To elucidate the mechanisms of immune evasion and to understand how the immune system interacts with glioma and stromal cells that shape the immune-suppressive landscape in GBM, there is an urgent need for immune-competent preclinical models that recapitulate the human disease. Human GBM is divided into three molecular subtypes (proneural:PR, classical:CL, and mesenchymal:MES) based on specific gene expression patterns and signature mutational profiles. Here, we report multi-dimensional analyses of six different transplantable mouse glioma models in the C57BL6/J background that represent all three human GBM molecular subtypes. We performed whole-exome sequencing as well as STR fingerprinting of each primary tumorsphere line and also performed immune phenotyping of each glioma model with flow cytometry. To gain molecular insights and determine cellular heterogeneity, we also performed single-cell RNA sequencing from the six models. Glioma cell analysis at the single-cell level revealed that cell-of-origin rather than the oncogenic driver (such as EGFRviii) plays a dominant role in determining the molecular phenotypes of glioma cells, driving their classification into a molecular subtype defined by human studies. In addition, we identified eight molecular subtypes of glioma-associated myeloid (GAMs), seven different subtypes of T cells, and provide molecular definitions of glioma-associated pericytes and endothelial cells in murine gliomas. In addition, we performed cross-species comparisons of glioma and immune cell subtypes between humans and mice at the single-cell level. Furthermore, we report qualitative and quantitative differences in the cell-to-cell communication among different stromal cells and glioma cells in each model, and propose that these interactions shape the local niche and functional neighborhoods. Finally, we leverage these preclinical models to elucidate underlying molecular mechanisms that drive differential sensitivities of each model to immunotherapies: anti-PD1, CTLA4, and 4-1BB in vivo. In summary, we report deeply characterized mouse models of human GBM subtypes and highlight their utility as preclinical models for immunotherapy evaluation and foundational tumor immunology studies. Citation Format: Caiyi Wang, Jose Maldonado, Thomas D. Gallup, Nourhan Abdelfattah, Jia-Shiun Leu, Nithin Joshy, joshy George, Jihye Paik, Massimo Squatrito, Kyuson Yun. Elucidating cell-to-cell communication and immunotherapy responses in deeply characterized mouse models of human glioma subtypes [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 1331.

Solid tumours display a limited response to immunotherapies. By contrast, haematological malignancies exhibit significantly higher response rates to immunotherapies as compared with solid tumours. Among several microenvironmental and biological disparities, the differential expression of unique immune regulatory molecules contributes significantly to the interaction of blood cancer cells with immune cells. The self-ligand receptor of the signalling lymphocytic activation molecule family member 7 (SLAMF7), a molecule that is critical in promoting the body's innate immune cells to detect and engulf cancer cells, is expressed nearly exclusively on the cell surface of haematologic tumours, but not on solid ones. Here we show that a bispecific nanobioconjugate that enables the decoration of SLAMF7 on the surface of solid tumours induces robust phagocytosis and activates the phagocyte cyclic guanosine monophosphate-adenosine monophosphate synthase-stimulator of interferon genes (cGAS-STING) pathway, sensitizing the tumours to immune checkpoint blockade. Our findings support an immunological conversion strategy that uses nano-adjuvants to improve the effectiveness of immunotherapies for solid tumours.