High Aldehyde Dehydrogenase Levels Are Detectable in the Serum of Patients with Lung Cancer and May Be Exploited as Screening Biomarkers.

Objectives: Since early detection improves overall survival in lung cancer, identification of screening biomarkers for patients at risk represents an area of intense investigation. Tumor liberated protein (TLP) has been previously described as a tumor-associated antigen (complex) present in the sera from lung cancer patients. Here, we set out to identify the nature of TLP to develop this as a potential biomarker for lung cancer screening. Materials and Methods: Beginning from the peptide epitope RTNKEASI previously identified from the TLP complex, we produced a rabbit anti-RTNKEASI serum and evaluated it in the lung cancer cell line A549 by means of immunoblot and peptide completion assay (PCA). The TLP sequence identification was conducted by mass spectrometry. The detected protein was, then, analyzed in patients with non-small cell lung cancer (NSCLC) and benign lung pathologies and healthy donors, by ELISA. Results: The anti-RTNKEASI antiserum detected and immunoprecipitated a 55\u2009kDa protein band in the lysate of A549 cells identified as aldehyde dehydrogenase isoform 1A1, revealing the molecular nature of at least one component of the previously described TLP complex. Next, we screened blood samples from a non-tumor cohort of 26 patients and 45 NSCLC patients with different disease stages for the presence of ALDH1A1 and global ALDH. This analysis indicated that serum positivity was highly restricted to patients with NSCLC (ALDH p < 0.001; ALDH1A1 p=0.028). Interestingly, the global ALDH test resulted positive in more NSCLC samples compared to the ALDH1A1 test, suggesting that other ALDH isoforms might add to the sensitivity of the assay. Conclusion: Our data indicate that ALDH levels are elevated in the sera of NSCLC patients, even with early stage disease, and may thus be evaluated as part of a marker panel for non-invasive detection of NSCLC

A perspective on the impact of radiation therapy on the immune rheostat.

The advent and success of immune checkpoint inhibitors (ICIs) in cancer treatment has broadened the spectrum of tumours that might be considered "immunogenic" and susceptible to immunotherapeutic (IT) intervention. Not all cancer types are sensitive, and not all patients with any given type respond. Combination treatment of ICIs with an established cytotoxic modality such as radiation therapy (RT) is a logical step towards improvement. For one, RT alone has been shown to be genuinely immunomodulatory and secondly pre-clinical data generally support combined ICI-RT approaches. This new integrated therapy for cancer treatment holds much promise, although there is still a lot to be learned about how best to schedule the treatments, manage the toxicities and determine what biomarkers might predict response, as well as many other issues. This review examines how RT alters the immune rheostat and how it might best be positioned to fully exploit IT

섬유증 및 종양 미세환경 내에서 활성화되도록 설계된 약물 전달 시스템 개발 연구

학위논문(박사) -- 서울대학교대학원 : 약학대학 약학과, 2023. 2. 이우인.질병 부위 내의 미세환경은 질병의 발달 및 치료에 중요한 역할을 담당한다. 질병 부위 내의 미세환경에서는 세포-세포간, 세포-환경 간의 복잡한 상호작용이 일어난다고 알려져 있다. 따라서 미세환경을 어떻게 조절하느냐에 따라서 치료의 성공 여부에 큰 영향을 끼친다. 약물 전달 시스템을 이용하면 국소적인 질병 부위에 특이적으로 약물을 전달할 수 있다. 본 박사학위 논문에서는 이러한 점에 착안하여 자극에 기반하여 활성화되는 약물 전달시슽템을 개발하고, 이를 통해 질병의 미세환경을 조절하여 질병 치료 효과를 보고자 하였다. 약물 전달 시스템에 이용한 자극 및 미세환경의 활용 방법에 따라서 총 세 가지 주제로 연구가 진행되었다. 첫 번째와 두 번째 주제는 종양 모델에서의 항암 치료 및 미세환경 조절에 관한 연구이며 세 번째 주제는 간섬유증 모델에서의 섬유성 미세환경 특이적인 항섬유화 펩타이드 전달에 관한 연구이다. 첫 번째 연구는 종양 치료 및 종양 미세환경의 면역 활성을 위한 적외선/원적외선 감응형 금 나노클러스터에 대한 연구이다. 두 종류의 빛에 동시에 감응하여 종양미세환경 내에서 광역학치료/광열치료 효과를 동시에 유도할 수 있었다. 또한 면역원성이 있는 CpG DNA 가닥을 금나노클러스터의 주형으로 활용함에 따라 수지상세포를 활성화하였다. 이에 따라 종양 특이적인 T 세포 반응을 유도함과 동시에 원발성 종양 진행을 억제할 수 있었다. 두 번째 연구는 저온 대기압 플라즈마 감응성 하이드로겔을 기반으로 한 면역조절 나노입자 및 종양 항원의 지속적 전달 시스템에 관한 연구이다. 저온 대기압 플라즈마에 의하여 종양 조직 내부에 종양 항원과 면역 조절 나노입자를 포함하는 하이드로겔이 형성된다. 종양 내에 형성된 이러한 네트워크는 수지상세포와 T 세포를 하이드로겔로 유도하고 항암 효과를 나타낼 수 있는 종양 미세면역환경을 형성한다. 세 번째 연구는 간섬유증 미세환경 내에서 특이적으로 높게 발현하는 효소가 있다는 것에 착안하여 개발된, 간섬유증 특이적 항섬유화 펩타이드 전달 시스템에 관한 연구이다. 간섬유화를 일으키는 활성화된 간성상세포를 미세환경의 특징을 이용하여 특이적으로 사멸시킬 수 있었으며, 미세환경 내에서 간성상세포의 비율만 낮출 수 있었다. Bile duct ligation, CCl4-induced fibrosis 그리고 지방간 기반 간섬유증까지 총 세 가지 간섬유화 모델에서 항섬유화 효과를 확인할 수 있었다. 이상의 연구들을 통하여 외부 자극을 통한 면역미세환경 조절 및 미세환경 내 내부 자극을 이용한 미세환경 조절을 약물전달시스템에 적용하였을 때 질병의 치료에 유의한 효과를 유도할 수 있다는 것을 확인할 수 있었다. 하지만 연구 별로 한계점이 존재하였다, 광열치료와 광역학치료에는 이를 유도할 수 있는 치료 물질이 포함되어야 하는데, 이 역시 임상적으로 사용되는 약물을 세심히 고를 필요가 있을 것으로 생각된다. 저온 플라즈마의 항암 치료에의 적용은 신규한 치료 방법이지만 그만큼 임상적으로 확인된 케이스가 적으므로 관련하여 더 많은 데이터를 축적할 필요가 있으며, 조직 내에서 플라즈마 기체가 작용할 수 있는 깊이가 깊지 않을 것이기 때문에 적용하고자 하는 종양의 크기가 치료 효과에 크게 영향을 끼칠 것으로 사료된다. 간섬유증 치료용 펩타이드의 전달은 치료 모델로서 간섬유화 모델을 이용했지만, 섬유아세포가 관여하는 다양한 섬유성 질환에 활용할 수 있을 것으로 사료된다. 다만 미세환경 내의 면역 세포 역시도 섬유증에서 중요한 역할을 담당하고 있다고 알려져있는데, 면역 세포의 변화를 평가하기 위해서는 추가적인 phenotype 연구가 필요하다. 본 연구에서는 흔하게 사용되는 페길화된 리포좀 제형을 사용하였으므로 제형 상 개선의 여지가 있으며, 세포를 표적화 할 수 있는 리간드와 함께 작용한다면 용량 대 약효를 더욱 증진시킬 수 있을 것이다. 질병의 미세환경은 복잡성을 띄고 있으며 질병의 종류 및 질병의 진행 정도에 따라, 그리고 개체 별로 큰 차이가 있을 수 있기 때문에 실제 임상에서는 효과가 어떻게 나타날지는 미지수이다. 또한 종양과 섬유성 질환 이외에도 미세환경 조절을 위한 약물전달 시스템을 개발하기 위해서는 더욱 넓은 범위의 연구가 필요하다. 따라서 자극 기반 약물전달시스템을 질병 미세환경에 적용하기 위해서는 질병 별로 보편성을 가지는 요소를 활용하여 질병에 적절한 자극을 선택하는 것이 중요할 것이다. 특히 저온 대기압 플라즈마를 포함한 외부 자극의 경우 대부분 피부 표면과 가까운 부위로 적용이 제한된다는 한계점이 있으므로 치료용 기기 등을 이용하여 이러한 한계를 극복하려는 노력이 필요하다. 여러 한계점에도 불구하고 미세환경의 조절과 국소적인 자극을 기반으로 한 약물전달 시스템의 시너지 효과는 주목할만한 잠재성을 가지고 있다. 특히 종양 및 섬유성 질환처럼 기존 치료법에 한계가 있는 질병에 대해서 본 연구에서 제시된 지극 기반 미세환경 조절용 약물 전달시스템은 유망한 치료 선택지가 될 수 있을 것이다.The microenvironment plays a crucial role in both the progression and treatment of diseases including tumor and hepatic fibrosis. However, current therapeutics are non-targeting or cell-targeting agents which can induce systemic adverse effects. The limited therapeutic efficacy of these agents was mainly associated with the deleterious microenvironment of the tissue. Therefore, strategies for targeted modulation of the microenvironment should be further investigated. Herein, we developed the programmed drug delivery systems that can be activated by exogenous or endogenous stimuli at the tumor and fibrotic microenvironment. This thesis work is composed of two parts, which are the exogenous stimuli-actuated delivery system for tumor immune microenvironment modulation and peptide-delivery nanotherapeutic for regulation of fibrotic microenvironment. In Part I, we developed stimuli-responsive delivery platforms that are programmed to be activated by light or cold atmospheric plasma (CAP). With these delivery systems, our goal was to eradicate primary tumors as well as distant tumors following immune microenvironment activation. In the first chapter, the gold-DNA nanocluster was developed for photo-immunotherapy on a tumor. Previous studies mainly used one light source for a single type of phototherapy, such as photothermal or photodynamic therapy, which has limited therapeutic efficacy as well as insufficient immune reaction. In this context, we designed gold-DNA nanocluster intercalating methylene blue for dual-light activated photo-immunotherapy. By exploiting a CpG sequence as a template for DNA polymer, delivery of nucleic acid to the dendritic cell was achieved to activate the tumor immune microenvironment followed by the prevention of distant tumor growth. Our in vivo study suggest that systemic injection of the nanocluster elicited enhanced tumor eradication and immune activation at the tumor site after the dual-light irradiation. In the second chapter, we developed programmed antigen-releasing hydrogel actuated by CAP irradiation. Previous clinical trials have utilized CAP for residual tumor removal, however, its limited penetration depth has limited its effectiveness in treating primary tumors. Our findings show that the CAP-activated hydrogel system is capable of sustained release of both tumor antigen and TGF-beta inhibitor, leading to increased anti-tumor immune response and primary as well as distant tumor eradication. These results highlight the potential of the CAP-responsive in situ hydrogel system as a promising therapeutic platform for overcoming the limitations of CAP's depth-related restriction in treating primary tumors. In part II of the thesis, the fibrotic microenvironment was modulated for the treatment of liver fibrosis. We designed an anti-fibrotic peptide delivery system that is programmed to be liberated by activated hepatic stellate cells (aHSCs). Despite the urgent global needs, there is no clinically approved therapeutic for liver fibrosis. In the fibrotic microenvironment, aHSCs play a central role in fibrogenesis and express distinct enzyme proteins such as fibroblast activation protein (FAP). Our goal was selective eradication of aHSCs in the fibrotic liver by exploiting promelittin, which is a FAP-responsive prodrug form of melittin. Treatment of promelittin-conjugated liposome could elicit anti-fibrotic effects in three different hepatic fibrosis models as well as apoptosis of aHSC. Although this peptide-delivery system was tested in liver fibrosis, it could be applied to other fibrotic diseases, such as lung fibrosis and cardiac fibrosis. Taken together, studies on programmed in situ drug delivery systems, which can be activated by stimuli, were achieved in this thesis work and showed promising therapeutic effects following the modulation of the tumor and fibrotic microenvironment. Furthermore, the drug delivery strategies suggested in this thesis address the current limitation of the therapeutics, which can broaden the current therapeutic indication.Abstract 1 Contents 3 List of Tables 5 List of Figures 6 Chapter I. Overview 8 I-1 Disease and the tissue microenvironment 9 I-2. Stimuli-responsive drug delivery systems 12 I-3. Scope of study 14 I-4. References 16 Chapter II. Photosensitizer-trapped gold nanocluster for dual light-responsive phototherapy 20 II-1. Introduction 22 II-2. Materials and methods 24 II-3. Results 28 II-4. Discussion 36 II-5. References 38 Chapter III. Cold plasma-actuated on-site hydrogel for remodeling tumor immune microenvironment 41 III-1. Introduction 42 III-2. Materials and methods 44 III-3. Results 53 III-4. Discussion 69 III-5. References 71 Chapter IV. Liver fibrosis-activated antifibrotic peptide delivery 75 IV-1. Introduction 76 IV-2. Materials and methods 77 IV-3. Results 85 IV-4. Discussion 98 IV-5. References 102 Chapter V. Conclusion 107 국문 초록 109박

New Perspectives on the Role of Vitiligo in Immune Responses to Melanoma

Melanoma-associated vitiligo is the best-studied example of the linkage between tumor immunity and autoimmunity. Although vitiligo is an independent positive prognostic factor for melanoma patients, the autoimmune destruction of melanocytes was long thought to be merely a side effect of robust anti-tumor immunity. However, new data reveal a key role for vitiligo in supporting T cell responses to melanoma. This research perspective reviews the history of melanoma-associated vitiligo in patients, the experimental studies that form the basis for understanding this relationship, and the unique characteristics of melanoma-specific CD8 T cells found in hosts with vitiligo. We also discuss the implications of our recent findings for the interpretation of patient responses, and the design of next-generation cancer immunotherapies

Tumor vaccination: Chitosan nanoparticles as antigen vehicles to promote tumor-directed T cell responses

Tumor vaccination is a promising approach for treatment of cancer. Tumor vaccines sensitize the immune system to tumor-specific antigens and thus enhance CD8+ T cell responses. This immune response’s potency can be increased by a strategy where nanoparticles act as vehicles leading to more efficient antigen uptake into antigen-presenting cells with chitosan emerging as a promising basic substance. In this study, the potential of antigen-loaded chitosan nanoparticles (CNPs) as delivery systems for inducing a potent CD8+ T cell response was assessed by using the model antigen SIINFEKL. First, uptake of FITC-conjugated antigen-loaded CNPs was verified. Small (approx. 200 nm in diameter) 90/10 CNPs did not show cytotoxic effects on human dendritic cells. Antigen-loaded CNPs did promote a more proinflammatory phenotype in murine and human dendritic cells. MHC-I mediated presentation of SIINFEKL on DC2.4 cells after treatment with SIINFEKL-loaded 90/10 CNPs was demonstrated. Coculturing CD8+ T cells isolated from spleens of OT-1 mice with DC2.4 cells that had been treated with SIINFEKL-loaded 90/10 CNPs led to elevation of activation marker expression on CD8+ T cells. Lastly, the functionality of these OT-1 derived CD8+ T cells activated by coculture with DC2.4 cells after pre-stimulation with 90/10 SIINFEKL CNPs was demonstrated by verifying CD8+ T cell-mediated antigen-specific lysis of PancOVA cells. Overall, the verification of internalization into dendritic cells, demonstration of low cytotoxicity and initiation of a more proinflammatory phenotype in dendritic cells, confirmation of MHC-I mediated antigen presentation and the activation of functionally active CD8+ T cells supports the hypothesis that CNPs are promising vehicles for tumor vaccination. Further studies have to be conducted to assess CNPs in a more clinical setting

Exploitation of the Toll-like receptor system in cancer: a doubled-edged sword?

The toll-like receptor (TLR) system constitutes a pylogenetically ancient, evolutionary conserved, archetypal pattern recognition system, which underpins pathogen recognition by and activation of the immune system. Toll-like receptor agonists have long been used as immunoadjuvants in anti cancer immunotherapy. However, TLRs are increasingly implicated in human disease pathogenesis and an expanding body of both clinical and experimental evidence suggests that the neoplastic process may subvert TLR signalling pathways to advance cancer progression. Recent discoveries in the TLR system open a multitude of potential therapeutic avenues. Extrapolation of such TLR system manipulations to a clinical oncological setting demands care to prevent potentially deleterious activation of TLR-mediated survival pathways. Thus, the TLR system is a double-edge sword, which needs to be carefully wielded in the setting of neoplastic disease

GUCY2C lysosomotropic endocytosis delivers immunotoxin therapy to metastatic colorectal cancer.

The emergence of targeted cancer therapy has been limited by the paucity of determinants which are tumor-specific and generally associated with disease, and have cell dynamics which effectively deploy cytotoxic payloads. Guanylyl cyclase C (GUCY2C) may be ideal for targeting because it is normally expressed only in insulated barrier compartments, including intestine and brain, but over-expressed by systemic metastatic colorectal tumors. Here, we reveal that GUCY2C rapidly internalizes from the cell surface to lysosomes in intestinal and colorectal cancer cells. Endocytosis is independent of ligand binding and receptor activation, and is mediated by clathrin. This mechanism suggests a design for immunotoxins comprising a GUCY2C-directed monoclonal antibody conjugated through a reducible disulfide linkage to ricin A chain, which is activated to a potent cytotoxin in lysosomes. Indeed, this immunotoxin specifically killed GUCY2C-expressing colorectal cancer cells in a lysosomal- and clathrin-dependent fashion. Moreover, this immunotoxin reduced pulmonary tumors \u3e80% (

Necroptosis in immuno-oncology and cancer immunotherapy

Immune-checkpoint blockers (ICBs) have revolutionized oncology and firmly established the subfield of immuno-oncology. Despite this renaissance, a subset of cancer patients remain unresponsive to ICBs due to widespread immuno-resistance. To "break" cancer cell-driven immuno-resistance, researchers have long floated the idea of therapeutically facilitating the immunogenicity of cancer cells by disrupting tumor-associated immuno-tolerance via conventional anticancer therapies. It is well appreciated that anticancer therapies causing immunogenic or inflammatory cell death are best positioned to productively activate anticancer immunity. A large proportion of studies have emphasized the importance of immunogenic apoptosis (i.e., immunogenic cell death or ICD); yet, it has also emerged that necroptosis, a programmed necrotic cell death pathway, can also be immunogenic. Emergence of a proficient immune profile for necroptosis has important implications for cancer because resistance to apoptosis is one of the major hallmarks of tumors. Putative immunogenic or inflammatory characteristics driven by necroptosis can be of great impact in immuno-oncology. However, as is typical for a highly complex and multi-factorial disease like cancer, a clear cause versus consensus relationship on the immunobiology of necroptosis in cancer cells has been tough to establish. In this review, we discuss the various aspects of necroptosis immunobiology with specific focus on immuno-oncology and cancer immunotherapy