摘要：通过改善 T 细胞浸润将冷肿瘤转变为热肿瘤-文献翻译-（2022-6-4）

通过改善 T 细胞浸润将冷肿瘤转变为热肿瘤

摘要

【1】以免疫检查点抑制剂（ICIs）为代表的免疫疗法极大地提高了恶性肿瘤治疗的临床疗效。ICI 介导的抗肿瘤反应依赖于能够识别和杀死肿瘤细胞的 T 细胞的浸润。【ICI-mediated antitumor responses depend on the infiltration of T cells capable of recognizing and killing tumor cells.】ICI 对“冷肿瘤”无效【ICIs are not effective in "cold tumors"】，其特征是缺乏 T 细胞浸润。为了充分发挥免疫疗法的潜力并解决这一障碍，了解 T 细胞浸润到肿瘤的驱动因素至关重要。我们对“冷肿瘤”潜在机制的理解进行了批判性审查【critical review】，包括 T 细胞启动受损【including impaired T-cell priming】和 T 细胞归巢到肿瘤床的缺陷。具有显著 T 细胞浸润的“热肿瘤”与更好的 ICI 疗效相关。在这篇综述中，我们总结了多种促进“冷肿瘤”转变为“热肿瘤”的策略，并讨论了这些策略导致 T 细胞浸润增加的机制。最后，我们讨论了纳米材料【nanomaterials】在肿瘤免疫治疗中的应用，并展望了这一新兴领域的未来。纳米药物和免疫疗法的结合增强了肿瘤抗原的交叉呈递，并促进了 T 细胞的启动【T-cell priming】和浸润。对这些机制的更深入了解为开发多种基于 T 细胞的联合疗法以提高 ICI 有效性开辟了新的可能性。

介绍

【2】最近，免疫检查点抑制剂（ICI），如纳武单抗和派姆单抗，已被应用于越来越多的癌症类型，形成了临床试验的范式【paradigm[ˈpærədaɪm]  n.范式;范例;典范;样式】治疗。尽管 ICI 已在多种肿瘤类型中显示出临床活性，但仍有相当一部分患者对 ICI 治疗没有反应。ICI 介导的抗肿瘤反应依赖于肿瘤中 PD-L1 的表达和能够识别和杀死肿瘤细胞的 T 细胞的浸润。CD8+ T 细胞等免疫细胞与癌症患者的生存期延长和免疫疗法疗效的提高有关 。肿瘤中缺乏 T 细胞会导致对免疫疗法的抵抗。嵌合抗原受体 (CAR，chimeric antigen receptor) T 细胞输注治疗白血病和淋巴瘤患者的成功也证明了 T 细胞在抗肿瘤免疫中的重要性 。考虑到癌症免疫治疗的潜在机制，肿瘤中 CD8+ T 淋巴细胞的浸润对于 ICI 的治疗反应很重要。

【3】根据细胞毒性免疫细胞在肿瘤微环境 (TME) 中的空间分布【spatial 空间的distribution】，将肿瘤分为三种基本免疫表型之一：免疫炎症、免疫排斥和免疫沙漠表型【immune-inflamed, immune-excluded and immune-desert phenotypes 】（图 1）。免疫炎症性肿瘤，也称为“热肿瘤”，其特征是高度【high】 T 细胞浸润、增加的干扰素-γ (IFN-γ) 信号传导、PD-L1 的表达和高肿瘤突变负荷 (TMB) 。具有炎症表型的肿瘤往往对 ICIs  更敏感。免疫排斥肿瘤和免疫沙漠肿瘤可被描述为“冷肿瘤”。在免疫排除的肿瘤中，CD8+ T 淋巴细胞仅存在于【localize only at】侵袭边缘，不能有效地浸润肿瘤 。在免疫沙漠肿瘤中，肿瘤及其周围不存在 CD8+ T 淋巴细胞。除了较差的 T 细胞浸润外，“冷肿瘤”还具有低突变负荷、低主要组织相容性复合物 (MHC，major histocompatibility complex) I 类表达，和低 PD-L1 表达的特点。免疫抑制细胞群，包括肿瘤相关巨噬细胞 (TAM) 和 T 调节细胞 (Tregs) 和髓源性抑制细胞 (MDSC)，也存在于冷肿瘤中。这些特征表明，冷肿瘤缺乏先天免疫，或者“冷肿瘤”中存在的先天抗肿瘤免疫特征可能由于免疫细胞的排斥而无效。与炎症表型相比，冷肿瘤很少对 ICI 单药治疗有反应。

肿瘤免疫表型。基于肿瘤微环境 (TME) 中 CD8+ T 淋巴细胞的空间分布，观察到三种免疫表型的梯度：免疫沙漠、免疫排斥和免疫炎症表型。在免疫沙漠表型中，肿瘤及其周围不存在免疫细胞。在免疫排斥表型中，免疫细胞聚集但不能有效浸润。在免疫炎症表型中，免疫细胞浸润但其作用受到抑制。值得注意的是，三种不同的表型对免疫检查点抑制剂的反应率不同。

【4】将 T 细胞驱动到 TME 是一个渐进的过程【gradual process 】（图 2）：肿瘤细胞死亡和抗原释放、抗原呈递细胞 (APC) 加工【processing】和肿瘤抗原的呈递，APC 和 T 细胞相互作用导致 T 细胞启动【 priming 】和激活。理想情况下，一旦被激活，这些肿瘤特异性 T 细胞就会离开【exit [ˈeksɪt]退场;出去;离去 】淋巴结并通过血流到达肿瘤部位。T细胞的产生及其与肿瘤细胞的身体接触对于抗肿瘤免疫的成功至关重要。一旦浸润瘤床，细胞毒性T淋巴细胞（CTL）特异性识别肿瘤细胞表面的抗原肽-MHC复合物，形成免疫突触【synapses美 [ˈsɪæpsɪz]  n.(神经元的)突触】，并释放穿孔素【perforin穿孔素;穿孔蛋白】和颗粒酶【granzyme】破坏肿瘤细胞。此外，CTLs通过Fas/FasL通路促进肿瘤细胞凋亡，并通过诱导铁死亡【 ferroptosis】和细胞焦亡【 pyroptosis细胞焦亡;细胞的炎症坏死;编程性细胞死亡;程序性细胞死亡;凋亡】抑制肿瘤。死亡的肿瘤细胞会释放额外的肿瘤抗原，从而放大 T 细胞反应。

肿瘤免疫循环和三种免疫表型。抗肿瘤免疫在很大程度上由 CD8+ T 淋巴细胞介导。肿瘤免疫循环由以下步骤组成：（1）肿瘤抗原释放，(2) 肿瘤抗原加工和呈递，(3) T 细胞启动和激活，(4) T 淋巴细胞通过血流运输至肿瘤，(5) T 淋巴细胞从脉管系统或肿瘤外周渗入肿瘤实质【tumor parenchyma】，(6) 识别肿瘤细胞，和 (7)细胞毒性 T 淋巴细胞 (CTL) 通过颗粒胞吐【granule exocytosis】作用或通过 Fas/FasL 途径破坏肿瘤细胞。死亡的肿瘤细胞会释放额外的抗原，从而使肿瘤免疫循环继续下去。值得注意的是，具有免疫沙漠表型（黄色）的肿瘤无法通过步骤 1-3，因为肿瘤及其边缘均不存在 T 淋巴细胞。由于肿瘤床缺乏 T 淋巴细胞，具有免疫排除表型（蓝色）的肿瘤不能超过步骤 4-5。由于 T 细胞衰竭和检查点激活，具有免疫炎症表型（红色）的肿瘤不能超过【 exceed】步骤 6-7。Adapted with permission from , copyright 2013 Elsevier.

【5】随着纳米技术的发展，基于纳米药物和生物材料的免疫治疗为未来提供了新的机遇。纳米药物在肿瘤治疗中具有独特的优势，例如提高药物精度和生物利用度以及减少免疫治疗引起的副作用。此外，纳米药物通过增强通透性和保留（EPR，enhanced permeability and retention）效应促进肿瘤中的选择性积累，或包含高亲和力配体以实现肿瘤的主动靶向。基于纳米技术的免疫疗法可增强肿瘤特异性免疫反应，促进 CTL 浸润，并抑制肿瘤转移和复发。

【6】鉴于 T 细胞浸润的重要性，了解 T 细胞归巢至肿瘤【homing to the tumor】的机制是必要的。为了提高免疫治疗的临床益处，ICI 可能与将“冷肿瘤”转化为“热肿瘤”的策略相结合，这可能使这些肿瘤对 ICI 治疗更加敏感。在这篇综述中，我们总结了 T 细胞浸润障碍的各种机制以及目前将 T 细胞引导至肿瘤的方法。最后，我们总结了基于纳米药物的局部治疗策略以增强 T 细胞浸润的最新进展、挑战和机遇，并讨论了该领域的进一步前景。

“冷肿瘤”表型的机制

【7】“冷肿瘤”的 ICI 反应率较低，其特征是没有 T 细胞浸润。在驱动 T 细胞进入肿瘤的过程中，有许多因素会影响 T 细胞启动和 T 细胞归巢至肿瘤床，从而导致 T 细胞无炎症表型和抗肿瘤免疫失败（图 3）。

三种不同肿瘤表型的机制。三种不同的表型与特定的生物学机制有关。具有免疫沙漠表型（黄色）的肿瘤可能由于缺乏肿瘤抗原、抗原加工和呈递机制缺陷或 DC-T 细胞相互作用受损而缺乏 T 细胞启动。具有免疫排除表型（蓝色）的肿瘤可能表现出致癌途径、异常趋化因子、异常脉管系统和缺氧或免疫抑制性肿瘤微环境（例如基质屏障）的激活。具有免疫炎症表型（红色）的肿瘤可以被许多免疫细胞浸润，但这些免疫细胞由于检查点激活而受到抑制。

ADO：腺苷；

ATP, adenosine triphosphate【三磷酸腺苷】; 

B2M: beta-2-microglobulin【β-2-微球蛋白】; 

BATF3: basic leucine zipper ATF-like transcription factor 3【碱性亮氨酸拉链ATF样转录因子3】; 

CAFs: cancer-associated fibroblasts【癌症相关成纤维细胞】; 

CRT, calreticulin【钙网蛋白】; 

CTLA4, cytotoxic T lymphocyte-associated antigen-4;【细胞毒性 T 淋巴细胞相关抗原 4】 

CXCL: CXC-chemokine ligand;【CXC-趋化因子配体】 

DNMT: DNA methyltransferase【DNA甲基转移酶】; 

ECM: extracellular matrix【细胞外基质】;

ETBR: endothelin B receptor【内皮素B受体】;

EZH2: enhancer of zeste homolog 2【zeste 同源物 2 的增强子】; 

FLT3L: Fms-like tyrosine kinase 3 ligand;【Fms样酪氨酸激酶3配体】 

GM-CSF: granulocyte-macrophage colony-stimulating factor;【粒细胞-巨噬细胞集落刺激因子】 

HDAC: histone deacetylase;【组蛋白脱乙酰酶】 

HEV: high endothelial venule;【高内皮小静脉】

HMGB1: high mobility family protein B1;【高迁移率家族蛋白 B1】 

ICAM: intercellular adhesion molecule;【细胞间粘附分子】 

IDO: Indoleamine 2,3-dioxygenase;【吲哚胺2,3-双加氧酶】 

IFN: interferon;【干扰素】

IL: interleukin;【白细胞介素】 

MDSC: myeloid-derived suppressor cell;【髓源性抑制细胞】 

MHC: major histocompatibility complex;【主要组织相容性复合体】 

PD-1, programmed cell death protein 1;【程序性细胞死亡蛋白1】 

PD-L1, PD-1 ligand;【PD-1配体】 

STC1: stanniocalcin 1【斯钙素1】; 

TAM: tumor-associated macrophage;【肿瘤相关巨噬细胞】 

TAP: transporter associated with antigen processing;【与抗原加工相关的转运蛋白】 

TGFβ: transforming growth factor-β;【转化生长因子-β】 

TIM3, T cell immunoglobulin and mucin domain-containing 3;【含 T 细胞免疫球蛋白和粘蛋白结构域 3】

TLR: Toll‑like receptor;【Toll样受体】 

TLS: tertiary lymphoid structure;【三级淋巴结构】 

TME: tumor microenvironment【肿瘤微环境】; 

Treg: T-regulatory cell;【T调节细胞】 

VCAM: vascular cell adhesion molecule;【血管细胞粘附分子】

VEGF: vascular endothelial growth factor.【血管内皮生长因子】

【8】T细胞启动障碍的最直接原因是由于缺乏肿瘤抗原而导致的T细胞识别不足。表 1 总结了 T 细胞启动缺陷的机制。一般来说，靶向肿瘤抗原可分为两大类：非突变自身抗原和非同义体细胞突变产生的新抗原。自身抗原包括在肿瘤细胞中异常表达或过表达的非突变蛋白，例如肿瘤相关抗原 (TAA) 和癌症/睾丸抗原 (CTAs，cancer/testis antigens)。虽然自身抗原也会引发【elicit】肿瘤免疫反应，但免疫反应的主要目标是新抗原，也称为肿瘤特异性抗原 (TSA)。新抗原对肿瘤细胞具有特异性，由癌症基因组中的体细胞突变产生。对肿瘤新抗原的识别可能会促进 T 细胞的启动和浸润，并可能导致长期的临床反应。

【导读

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【9】TMB 被广泛表征为【characterized as】肿瘤中非同义【nonsynonymous】单核苷酸【 single-nucleotide】突变的总数。一般来说，具有较高 TMB 的肿瘤被认为携带较高的新抗原负荷，可以被 T 细胞识别，使它们更有可能引发【 prime事先指点;使(某人)做好准备;把(事物)准备好】免疫系统。据报道，在多种肿瘤类型中，高 TMB 与对 ICI 的反应改善之间存在显著关联。TMB 已被用作预测程序性细胞死亡蛋白 1 (PD-1) 抑制剂疗效的新型生物标志物。与 ICI 功效的重要性一致，高 TMB 与更大的免疫细胞浸润有关。此外，一项多组学【mutliomics 多元组学】网络分析显示，在以反复突变【recurrent mutations】为特征的肿瘤中，例如黑色素瘤和结直肠癌，突变或新抗原负荷与 CTL 浸润呈正相关。考虑到高 TMB 和肿瘤特异性 T 细胞之间的关系，低突变负荷或新抗原负荷在一定程度上有助于缺乏免疫浸润和获得 ICI 抗性。然而，在以复发性拷贝数改变为特征的肿瘤中，例如乳腺癌，肿瘤特异性 T 细胞浸润和新抗原负荷【neoantigen load】之间缺乏相关性。对癌症基因组图谱 (TCGA) 中 266 个黑色素瘤数据的调查显示，冷肿瘤和热肿瘤之间的抗原表达没有差异。这一发现表明，除了涉及低 TMB 的机制外，其他机制也有助于 T 细胞浸润的缺失。

【10】在识别肿瘤抗原后，APCs 处理抗原并在其表面表达相应的抗原肽-MHC I 类复合物。然而，APM 的改变，例如 MHC-I 分子表达的下调或 β-2-微球蛋白 (B2M) 的缺失，存在肿瘤抗原时会限制抗原肽-MHC I 类复合物的呈递。在抗原处理和呈递过程中，与抗原处理相关的转运体（TAP）将胞浆裂解【cytosolic cleaved】抗原转运至内质网【endoplasmic reticulum】，以与MHC结合。TAP 中的缺失【The deletion in TAP】与抗原呈递过程中的缺陷有关，这进一步影响了 T 淋巴细胞的启动。B2M 是 MHC 的不变链【invariant chain】，对于 MHC-I 成功折叠【successful folding】和运输到细胞表面至关重要。敲除 M202 和 M233 人黑色素瘤细胞系中的 B2M 基因导致其表面不表达 MHC-I 分子，并且不存在肿瘤特异性 T 细胞识别和细胞毒性。【and the absence of tumor-specific T-cell recognition and cytotoxicity .】在 B2M 基因敲除小鼠肺癌模型中观察到了相同的【Identical完全相同的】结果，该模型显示出对 PD-1 阻断的抗药性。此外，溶酶体途径【lysosomal pathway】与 CD8+ T 淋巴细胞浸润减少有关。在胰腺导管腺癌 (PDAC) 中，自噬相关受体【autophagy-associated receptor】 NBR1 诱导肿瘤细胞表面的 MHC-I 降解，进而影响 T 细胞反应。这些发现表明，肿瘤抗原加工和呈递途径的缺陷会抑制 T 细胞启动和癌症免疫疗法的有效性。

【11】树突状细胞 (DC，Dendritic cells) 是专业的 APC，具有获取抗原、迁移到次级淋巴器官（例如淋巴结和脾脏）和启动体内免疫反应的独特能力。DC 激活需要其表面的模式识别受体 (PRRs，pattern recognition receptors ) 识别“危险信号”，包括病原体相关分子模式 (PAMPs，pathogen-associated molecular patterns) 和损伤相关分子模式 (DAMPs，damage-associated molecular patterns )。这种识别使 DC 在与 T 细胞接触时能够将肿瘤抗原肽-MHC I 类复合物呈递给 T 细胞。DC 还表达共刺激信号，如 B7（包括 CD80 和 CD86），提供 T 细胞激活所需的次级信号。肿瘤细胞可以通过捕获“危险信号”来介导 DCs 吞噬作用的减弱。例如，细胞内检查点斯钙素 1 (STC1，stanniocalcin 1) 可以捕获 DAMP（例如钙网蛋白 (CRT，calreticulin )）并抑制 DC 吞噬作用和 T 细胞活化，从而有助于肿瘤免疫逃逸。此外，STC1 与黑色素瘤患者的低 T 细胞活化和较差的存活率有关。

【12】DC 通常分为两大类：以产生 IFN-α 为特征的浆细胞样 DC (pDC，plasmacytoid DCs) 和有效刺激 T 细胞增殖的常规 DC (cDC，conventional DCs)。CDC 进一步分为两个不同的子集：BATF3 依赖性 DC 和 IRF4 依赖性 DC。BATF3 DCs 能够通过 MHC-I 途径交叉呈递肿瘤来源的抗原，从而启动 T 细胞。此外，BATF3 DC 是 CXC 趋化因子配体 9 (CXCL9，CXC-chemokine ligand 9) 和 CXCL10 的主要来源，这两种关键趋化因子需要将表达 CXCR3 的 CD8+ T 细胞募集到肿瘤中。BATF3 DC 标志物（例如，BATF3 和 IRF8）、CXCL9、CXCL10 和 CXCL11 的表达与黑色素瘤中的 CD8+ 效应 T 细胞表型之间存在显着相关性。在没有 BATF3 DCs 的情况下，CD8+ 效应 T 细胞不能迁移到肿瘤中，因此抗肿瘤免疫是有缺陷的。这一发现验证了 BATF3 DCs 可能对于引发和募集对抗肿瘤所必需的内源性 T 细胞至关重要的观点。

【13】Fms 样酪氨酸激酶 3 配体 (FLT3L，Fms-like tyrosine kinase 3 ligand ) 和粒细胞-巨噬细胞集落刺激因子 (GM-CSF，granulocyte-macrophage colony-stimulating factor ) 的调节对于 DCs 的分化和募集很重要。FLT3L 是一种生长因子，可促进造血祖细胞【hematopoietic progenitor cells】从骨髓分化为 DC 谱系。肿瘤来源的 FLT3L 增加了小鼠肿瘤中 BATF3 DC 和 CD8+ T 淋巴细胞的浸润，并增强了引流淋巴结 (DLN，draining lymph nodes) 中的迁移和驻留 DC 亚群，表明 FLT3L 对 DC 细胞具有动员作用。FLT3L 或 GM-CSF 的缺乏导致次级淋巴器官中 DCs 数量减少和 T 细胞免疫反应减弱。鉴于 DC-T 细胞串扰在初始 T 细胞【naïve T-cell 】启动中的重要作用，DC 激活受损、DC 缺乏以及共抑制信号的过度表达可导致 T 细胞激活受损。

【14】出于本综述的目的，我们总结了阻止 T 细胞归巢到肿瘤床的机制（表 2）。越来越多的证据表明，肿瘤细胞致癌途径的激活与“冷肿瘤”表型和免疫治疗耐药性有关。在 WNT/βcatenin 阳性黑色素瘤肿瘤中，CCL4 的产生减少导致 BATF3 DC 向 TME 的募集减少。最终，在没有 BATF3 DC 产生的 CXCL9 和 CXCL10 的情况下，CTL 不会被招募到肿瘤中。对人类转移性黑色素瘤样本的分析显示，CD8A 表达与 β-连环蛋白【β-catenin】信号通路的激活呈负相关。直接注射 BATF3 DC 有助于恢复 β-连环蛋白阳性肿瘤中的 T 细胞浸润并导致适度的肿瘤抑制【resulted in modest tumor suppression . 】。这一结果表明，WNT/β-连环蛋白信号激活和有缺陷的 BATF3 DC 募集介导了 T 细胞排斥和肿瘤细胞从免疫系统中逃逸。值得注意的是，只有当 β-连环蛋白位于细胞核中时，这种致癌途径的激活才会排斥【excludes】 CTL。这一发现表明，CTLs 的排斥机制与 β-catenin 特异性诱导的转录程序有关。此外，只有 48% 的“冷”黑色素瘤表现出活跃的 βcatenin 信号传导，这表明其他致癌途径可能介导免疫排斥【 immune exclusion】。

【15】PTEN 的缺失激活了 PI3K/AKT 通路，这与黑色素瘤的非炎症性 T 细胞表型和免疫抵抗性【 immune resistance】有关。已发现 PTEN 表达的丧失会降低自噬体蛋白 【autophagosome protein】LC3 的脂化【lipidation】，导致自噬活性降低，从而抑制 T 细胞启动和 T 细胞介导的抗肿瘤反应。与表达 PTEN 的肿瘤相比，PTEN 缺陷型黑色素瘤中的 CD8+ T 细胞浸润显着减少。TCGA 数据集分析的结果表明，在 PTEN 低表达的黑色素瘤中，T 细胞效应分子（例如，IFN-γ 和颗粒酶 B）的表达显着降低。

【16】作为与癌症进展相关的最常见突变的基因，RAS可以导致多种信号通路的激活，例如MAPK和PI3K，从而驱动肿瘤发生。此外，致癌 K-RAS 突变介导炎症和与 TME 的串扰。例如，致癌 K-RAS 突变通过产生抑制性细胞因子（例如，IL-6 和 IL-8）、激活 NLRP3 炎性体【inflammasome】和释放趋化因子（例如，CCL5 和 CCL9）来诱导促肿瘤炎症【induce tumor-promoting inflammation】。

【17】此外，通过 MYC 的致癌信号传导增强了肿瘤细胞上 CD47 和 PD-L1 的表达。CD47与巨噬细胞、DC等APCs表面的抑制性受体信号调节蛋白-α（SIRPα）结合，可阻止肿瘤细胞的吞噬作用，干扰抗原的摄取。致癌 KRAS 和 MYC 协同诱导免疫调节。例如，小鼠肺癌模型中 KRAS 和 MYC 的共激活导致 CCL9 和 IL-23 的产生。这介导基质重编程，促进血管生成，并从肿瘤中排除 T 和 B 细胞和 NK 细胞。还发现，在临床前小鼠模型中，非小细胞肺癌 (NSCLC) 中 LKB1 的失活突变与中性粒细胞增加和 T 细胞浸润减少有关。此外，CDK4/6 和 STAT3 激活与非炎症性 T 细胞表型相关。总之，这些结果表明，致癌途径的激活不仅可以影响肿瘤细胞，还可以影响 T 细胞介导的抗肿瘤免疫。

【18】效应T淋巴细胞上的一些趋化因子受体【chemokine receptors】与相应趋化因子之间的相互作用可能影响效应T淋巴细胞向肿瘤部位的运输。据报道，缺乏几种趋化因子，包括 CXCL9、CXCL10、CCL4、CCL5、CXCL16 或 CX3CL1，会导致 T 细胞排斥。考虑到 TH1 型趋化因子 CXCL9 和 CXCL10 对 T 细胞募集的重要性，某些肿瘤表现出低水平的 CXCL9 和 CXCL10 表达，这可能解释了效应 T 淋巴细胞向这些肿瘤床的浸润减少。例如，BATF3 DCs 是 CXCL9 和 CXCL10 的主要来源，BATF3 DCs 的缺乏导致 CXCL9 和 CXCL10 的低表达。此外，肿瘤中的表观遗传调控【epigenetic regulation】对于维持这些细胞因子的低表达水平也很重要。DNA 甲基转移酶 (DNMT) 和 zeste 同源物 2 增强子 (EZH2，enhancer of zeste homolog 2 ) 可分别介导 DNA 甲基化和组蛋白赖氨酸甲基化，从而抑制卵巢癌中 CXCL9 和 CXCL10 的表达。类似的结果已在结肠癌中得到证实。在临床前模型中，表观遗传调节剂治疗促进了效应 T 细胞的肿瘤浸润并增强了抗 PD-L1 的作用。除 CXCL9 和 CXCL10 外，CCL5 表达与 CD8+ T 细胞浸润呈正相关。CCL5 与 CCR5 的结合促进了 CD8+ T 细胞的募集。然而，DNA 甲基化导致 CCL5 表达缺失，进而导致 CD8+ T 细胞浸润缺失。在 NSCLC 小鼠模型中，DNMT 抑制剂和组蛋白去乙酰化酶 (HDAC) 抑制剂的联合使用增加了内源性逆转录病毒 (ERV) 的表达，进而诱导 I 型 IFN 反应。这种联合治疗通过下调致癌 MYC 信号转导逆转 NSCLC 模型的免疫抗性，导致 CCL5 增加并促进 T 细胞浸润到肿瘤中。

【19】然而，一些趋化因子不利于【detrimental】 T 细胞向肿瘤床的运输。基质细胞，尤其是癌症相关成纤维细胞 (CAF)，是 CXCL12 的主要生产者。CAFs 产生的 CXCL12 将 CTLs 误导【misdirects】到瘤外基质并阻止 CTLs 进入肿瘤。此外，据报道，CXCL8 表达升高与肿瘤中 T 细胞数量减少、中性粒细胞和单核细胞浸润增加以及对 ICI 的反应有限有关。这些结果揭示了趋化因子受体和配体相互作用对 CTL 归巢至肿瘤及其与 TME 整合的调节作用。

【20】肿瘤床中足够的 T 细胞浸润不仅取决于适当趋化因子的募集，而且还受肿瘤脉管系统的控制。在将 CD8+ T 淋巴细胞运输至肿瘤期间，它们必须进入肿瘤循环系统，粘附于血管内皮细胞并迁移穿过血管壁。CD8+ T 细胞向肿瘤的募集需要血管内皮粘附分子的作用，包括 P-和 E-选择素【selectin】、细胞间粘附分子 (ICAM， intercellular adhesion molecules) 和血管细胞粘附分子 (VCAM，vascular cell adhesion molecules)。然而，肿瘤内皮细胞上粘附分子的下调或无效聚集【downregulation or ineffective aggregation】会导致内皮细胞无能【anergy】，并减少效应 T 细胞向肿瘤部位的运输。内皮素与内皮细胞上的相应受体内皮素 B 受体 (ETBR，endothelin B receptor ) 结合，并减少 ICAM-1 的产生，从而抑制 CD8+ T 细胞与内皮细胞的粘附。此外，由肿瘤和基质细胞产生的血管内皮生长因子 (VEGF) 可刺激内皮细胞增殖，导致新血管形成，通常伴有组织灌注受损和血管通透性增加。VEGF 还降低内皮细胞表面重要分子（如 VCAM-1）的表达，最终阻止 T 细胞迁移到 TME。肿瘤内皮细胞抑制 T 细胞迁移的另一种机制是调节免疫细胞活性或活力【activity or viability】。IL-10、前列腺素 E2 (PGE2) 和 VEGF 诱导肿瘤内皮细胞中的 FasL 上调以杀死肿瘤相关的 T 细胞，而抗 FasL 减弱了【attenuated】这种杀伤作用。抑制 COX 和 PGE2 活性的乙酰水杨酸 (ASA) 和抗 VEGF 抗体可促进 TME 中 CD8+ T 淋巴细胞浸润并改善预后。此外，周细胞【pericyte】异常和覆盖不足【inadequate coverage】阻碍了内皮细胞完整性的维持，导致肿瘤脉管系统的功能失调渗漏和流动特性。然而，其他结构可以促进 CD8+ T 淋巴细胞从血管转移到肿瘤部位。高内皮小静脉 (HEV，high endothelial venules) 和相关三级淋巴结构 (TLS) 的形成有助于 T 细胞迁移到 TME，并且通常与更好的预后相关。

【21】此外，血管紧密连接受损和通透性增加促进缺氧、酸中毒和坏死，从而抑制免疫效应 T 细胞功能和抗肿瘤免疫。作为癌症的标志，缺氧是由肿瘤细胞增殖引起的氧气需求增加和血管生成引起的血液供应不足引起的。缺氧诱导因子 1 (HIF1，Hypoxia-inducible factor 1 ) 是缺氧激活的关键转录因子。缺氧以多种方式抑制 T 细胞浸润。首先，缺氧促进免疫抑制细胞向 TME 的募集。其次，缺氧诱导的 CCL28 和 VEGF 促进血管生成并影响 T 细胞运输。最后，两种外核苷酸酶 CD39 和 CD73 的表达可以在肿瘤中上调以响应缺氧和转化生长因子-β (TGFβ)。CD39 和 CD73 催化 ATP 依次转化为细胞外腺苷 (ADO， adenosine) 。ADO 与腺苷 A2A 受体 (A2AR) 结合并抑制细胞因子如 IL-2 的产生和 T 细胞的发育和增殖。A2AR 的抑制增加了 T 淋巴细胞浸润并导致小鼠黑色素瘤模型中的肿瘤控制得到改善，这表明 ADO 信号通路在促进 T 细胞排斥中具有潜在作用。此外，ADO 可通过抑制 NK 细胞和 DCs 的效应功能，促进 MDSCs 和 Tregs 的募集和极化来削弱抗肿瘤免疫。

【22】肿瘤部位的免疫抑制微环境，包括致密的基质和免疫抑制细胞和因子，在“冷肿瘤”中可以防止 T 细胞启动和浸润。TGFβ 是一种有效的免疫抑制细胞因子，可促进免疫逃逸并阻止 TH1 效应表型的获得。TME中丰富的CAF是TGFβ的主要生产者。CAF 产生的 TGFβ 增加与肿瘤排除 T 细胞和对atezolizumab 反应差有关。TGFβ 通过抑制 IL-2 的产生来限制 CD4+ T 淋巴细胞的增殖并诱导幼稚【naïve】 CD4+ T 淋巴细胞转化为 Treg。TGFβ 还会对 DC 分化和抗原呈递功能产生负面影响，从而干扰 T 细胞启动。总之，TGFβ通过影响T细胞分化和功能，阻止T细胞浸润到肿瘤中来阻碍抗肿瘤免疫。

【23】色氨酸代谢通常在广泛的癌症中失调，并与免疫抗性有关。肿瘤细胞中的吲哚胺 2,3-双加氧酶 (IDO) 将必需氨基酸色氨酸转化为犬尿氨酸【kynurenine】，从而阻断 T 淋巴细胞的启动并促进 Treg 的发育。IDO 还募集和激活 MDSC，并抑制肿瘤特异性 T 淋巴细胞在肿瘤中的积累。IDO 抑制剂如 epacadostat 和 navoximod 已与 ICI 联合使用，在临床试验中取得了可喜的结果。但epacadostat联合pembrolizumab在III期临床ECHO-301研究中的失败表明靶向IDO的药物的有效性需要进一步考虑。

【24】CAFs是肿瘤基质中的关键细胞成分，可以促进肿瘤生长。CAF主要位于肿瘤的浸润边缘，通过合成和重塑细胞外基质（ECM）和产生细胞因子，将肿瘤边缘转化为免疫“冷”区，调节肿瘤转移和影响血管生成。CAF 通过多种机制导致免疫抑制和 T 细胞排斥。首先，CAF 产生细胞外基质，形成物理屏障以防止 T 细胞浸润到肿瘤区域。其次，CAF 产生的 CXCL12 已被证明可抑制胰腺癌模型中肿瘤内的 T 淋巴细胞浸润。第三，CAFs 还可以通过产生 TGFβ 和 IL-6 来降低 T 细胞反应并发挥免疫抑制作用。重新编程 CAF 是“规范化”【normalize】TME 的有效策略。这种策略降低了 ECM 水平，减压血管，并增加了 T 细胞的渗透程度，以改善癌症治疗。

【25】此外，TAMs 通过调节 ECM 和介导 CCL2 和 CCL5 的硝化【 nitration】将 T 细胞排除在肿瘤之外。TAM 通过产生 VEGF 和基质金属蛋白酶 9 (MMP9，matrix metalloproteinase-9) 促进异常血管生成，从而影响 T 细胞募集。细胞因子集落刺激因子 1 (CSF-1，Cytokine colony-stimulating factor-1) 和 CSF-1R 相互作用能够促进骨髓细胞分化为免疫抑制性 M2 巨噬细胞表型。用 CSF1R 抑制剂靶向 TAM 可减少 TAM 的数量并增加效应淋巴细胞（如 CD8+ T 细胞）的浸润。

【26】肿瘤细胞的典型特征是高速率的葡萄糖摄取和活跃的糖酵解，即使在氧气存在的情况下也是如此。这种现象被称为“Warburg 效应”。在这个过程中，葡萄糖被迅速消耗，TME 中的乳酸含量增加。缺乏葡萄糖、富含乳酸的 TME 对浸润的 T 细胞施加代谢压力，导致局部免疫抑制和 ICI 抗性。TME 中的葡萄糖剥夺通过代谢介导 T 细胞低反应性，抑制 mTOR 活化，并降低糖酵解能力和 IFN-γ 产生。此外，糖酵解活性和 T 细胞浸润在多种肿瘤中呈负相关。与这一观察结果一致，肾细胞癌中的高葡萄糖转运蛋白 1 (GLUT-1) 表达与 CD8+ T 细胞的低浸润有关。这些结果表明糖酵解 (Warburg) 肿瘤与非炎症 T 细胞表型有关。有趣的是，除了肿瘤细胞外，CAF 和 TAM 等基质细胞也可以通过所谓的“反向 Warburg 效应”促进 TME 中的乳酸积累。靶向肿瘤和基质细胞中的葡萄糖代谢和乳酸产生，例如抑制 LDH-A，可能是促进 T 细胞浸润的有效策略。TME 的乳酸积累和酸化抑制抗肿瘤免疫。乳酸诱导的酸中毒会损害单核细胞向 DC 的分化并抑制 DC 的抗原呈递功能，进而抑制 T 细胞活化。TME 中的高浓度乳酸和酸化可抑制单羧酸转运蛋白 1 (MCT1，monocarboxylate transporter 1 ) 介导的 T 细胞释放乳酸，并抑制利用有氧糖酵解的 T 细胞增殖。此外，乳酸抑制CTLs的趋化性和抗肿瘤活性，促进肿瘤免疫逃逸。抑制乳酸产生或恢复 TME 的生理 pH 值可以逆转乳酸对抗肿瘤免疫的抑制作用。例如，用碳酸氢钠中和肿瘤酸度并结合 ICI 或过继细胞疗法 (ACT) 可以有效促进 T 细胞浸润并改善各种小鼠肿瘤模型中的抗肿瘤反应。

【27】除了葡萄糖，肿瘤和免疫细胞之间的代谢竞争还包括氨基酸和脂肪酸。例如，肿瘤中高胆固醇酯化率会抑制 T 细胞受体 (TCR) 聚集和免疫突触形成。胆固醇酯化【cholesterol esterification】关键酶ACAT1抑制剂avasimibe能促进CD8+T细胞增殖，表现出良好的抗肿瘤作用。新的研究还证实，抑制 PCSK9（一种调节胆固醇代谢的关键蛋白）可上调肿瘤细胞表面的 MHC-I 水平，增加 CTL 的肿瘤内浸润，并与抗 PD1 抗体协同抑制肿瘤生长。考虑到肿瘤代谢和免疫细胞代谢之间的相互作用，引导代谢途径以减少对 T 细胞的代谢压力是提高免疫治疗疗效的有希望的策略。

【28】ICI 通过激活基于 T 细胞的抗肿瘤免疫，彻底改变了癌症治疗。然而，由于介导 T 细胞排斥的多种机制，大量患者对 ICI 反应不佳。几种方法已被证明可以将 T 细胞驱动到肿瘤中。这些方法“激发”【fire up】“冷肿瘤”以提高 ICI 的疗效（图 4 和表 3）。

将“冷肿瘤”转变为“热肿瘤”的方法。

此处重点介绍了一些导致 T 细胞浸润增加和免疫检查点抑制剂疗效提高的代表性方法。(A) 溶瘤病毒【Oncolytic viruses】、局部热消融疗法（例如射频消融）、化学疗法和放射疗法都能够诱导免疫原性细胞死亡（ICD，immunogenic cell death ）以促进 T 细胞的启动和活化。局部施用免疫佐剂如 TLR 激动剂可促进树突状细胞 (DC) 的活化。表观遗传修饰抑制剂可以通过增加肿瘤抗原的表达和恢复抗原加工和呈递机制来促进 T 细胞启动。(B) 癌症疫苗和过继性细胞疗法，例如 CAR-T 细胞，可以促进肿瘤特异性 T 淋巴细胞的扩增。(C) 内在致癌途径抑制剂、表观遗传修饰抑制剂、抗血管生成疗法、TGFβ抑制剂和 CXCR4 抑制剂促进 T 细胞运输并使 T 细胞更有效地浸润肿瘤。

【29】先天免疫传感通路【Innate immune sensing pathways】在抗肿瘤免疫的发展中发挥着关键作用。PRR 家族包括 Toll 样受体 (TLR，Toll-like receptors)、NOD 样受体 (NLR，NOD-like receptors)、RIG-I 样受体 (RLR，RIG-I-like receptors) 和 C 型凝集素受体 (CLR，C-type lectin receptors)。当 TLR 受到刺激时，DC 可以产生多种促炎细胞因子，包括肿瘤坏死因子 (TNF)、IL-1 和 I 型 IFN。DCs是I型干扰素的主要来源，它促进MHC-I在肿瘤细胞表面的表达和DCs的成熟，从而促进T细胞的启动。

【30】与疫苗和 CAR-T 细胞等疗法相比，免疫佐剂利用肿瘤中的内源性抗原库，并已被用于增强免疫反应以治疗恶性肿瘤。TLR7/8 激动剂咪唑喹啉与偶联纳米颗粒局部给药可显着激活次级淋巴器官的 DC，上调其表面 MHC-II、CD40 和 CD86 的表达，并增加肿瘤特异性 CD8+ T 淋巴细胞的数量，从而抑制肿瘤生长。在晚期恶性黑色素瘤患者的临床试验中，TLR9 激动剂 SD-101 和派姆单抗联合治疗导致 I 型 IFN 产生和 CD8+ T 细胞浸润增加，并可能提高临床疗效。

【31】作为细胞质中的 DNA 受体，DC、巨噬细胞和其他免疫细胞中的环状 GMP-AMP 合酶 (cGAS， cyclic GMP-AMP synthase) 识别细胞质中的异常 DNA 并催化 cGAMP 的形成，从而激活 STING 信号通路。STING 信号通路的激活以 TBK1-IRF3 依赖性方式介导促炎细胞因子【proinflammatory cytokines】（如 I 型 IFN）和趋化因子（如 CXCL10）的表达，从而启动抗肿瘤免疫反应。全身性 cGAMP 模拟物 SR-717 激活 STING 信号通路，促进 CD8+ T 细胞、NK 细胞和 CD8α+ DC 活化，并显着抑制肿瘤生长。SR-717 还以 STING 依赖性方式诱导 PD-L1 表达，揭示了 STING 激动剂和 ICI 联合用于肿瘤治疗的意义。在对 PD-1 阻断反应低的小鼠肿瘤模型中，PD-1 阻断和 STING 激动剂 MSA-2 的组合增加了肿瘤 CD8+ T 淋巴细胞的浸润，更好地抑制了肿瘤生长。

【32】OVs 现在被认为是具有强大抗癌活性的新兴疗法。除了选择性肿瘤溶解外，它们还可以激活先天性【 innate】和适应性【adaptive】免疫反应，从而导致 TME 的改变。首先，OVs 对肿瘤细胞的裂解诱导免疫原性细胞死亡 (ICD，immunogenic cell death )，导致细胞内 TAA、PAMP 和 DAMP 的大量释放。ICD 期间会释放三种 DAMP：被动释放的高迁移率家族蛋白 B1 (HMGB1，high mobility family protein B1)、主动分泌的细胞外 ATP 和细胞表面表达的 CRT。这些 DAMP 作为佐剂促进 DC 摄取并将肿瘤抗原交叉呈递给 DLN 中的 T 淋巴细胞。OVs 还通过刺激 I 型 IFN 的产生来改善 DCs 的功能。免疫佐剂与肿瘤残留物中的肿瘤抗原相互作用，并作为个体化原位疫苗促进 T 细胞启动。

其次，OVs 刺激 CXCL9 和 CXCL10 的产生并上调选择素【selectins】和整合素【 integrins】的表达，为 T 细胞运输提供关键信号。此外，OVs 对 ECM 的降解破坏了 T 细胞浸润的物理屏障。OVs 还可以耗尽【deplete大量减少;耗尽;使枯竭】 CAFs、TAMs 和 MDSCs 的免疫抑制作用，显着改变 TME。

【33】Talimogene laherparepvec (T-VEC) 首次被证明可有效作为黑色素瘤的溶瘤病毒疗法。T-VEC 和 pembrolizumab 的联合治疗增加了晚期恶性黑色素瘤患者的 CD8+ T 淋巴细胞浸润、IFN-γ 表达和 PD-1 阻断的治疗效果。柯萨奇病毒【coxsackievirus】和派姆单抗的联合治疗也观察到了类似的效果。这些研究表明，OVs 和 ICIs 的联合治疗可以改善 CD8+ T 细胞的浸润和活化，并有助于克服癌症对 ICIs 的耐药性。结合 T 细胞治疗，促进 T 细胞增殖和浸润到局部 TME 是 OVs 发展的一个潜在方向。

【34】以前认为化学疗法和放射疗法通过直接杀死肿瘤细胞来发挥其抗肿瘤作用。然而，积累的证据表明，通过化学疗法和放射疗法抑制肿瘤也依赖于刺激免疫系统。当对局部肿瘤进行放射治疗时，照射野外的远处肿瘤也会缩小。这种现象被称为“异位效应”【abscopal effect】，表明免疫系统在放射治疗介导的抗肿瘤反应中的重要性。放射治疗对肿瘤细胞造成损伤后，ROS 和内质网 (ER， endoplasmic reticulum ) 应激介导细胞应激并导致 ICD。这种级联反应促进 DC 活化，增加 TNFα 和 IL-1 的产生，并在体内产生内源性癌症疫苗。放射治疗后，内皮细胞表达 ICAM1、VCAM1 和 E-选择素，从而促进免疫细胞的吸引。辐射还通过诱导肿瘤细胞表达和释放趋化因子（例如，CXCL10和CXCL16）来促进效应T淋巴细胞向肿瘤部位的运输。然而，考虑到放射治疗的不良反应，有必要优化放射治疗期间的放射剂量和分割水平。单个剂量小于 8-10 Gy 的分次放疗有助于诱导足够的 ICD，而不会增加缺氧或免疫抑制，从而诱导从头抗肿瘤反应。【inducing a de novo antitumor response .】在临床前研究中，立体定向放射治疗 (SBRT) 增加了肿瘤和 DLN 中的效应 T 细胞浸润，并与更高的存活率相关。

【35】除放疗外，许多化疗药物还可以通过增强免疫原性和增加 T 细胞浸润来发挥免疫刺激作用。ICD 诱导化疗已在各种小鼠模型中显示，有助于响应 ICI 将“冷肿瘤”转变为“热肿瘤”。降低全身化疗毒副作用并增强化疗药物诱导的免疫原性的新免疫疗法值得进一步探索。一种新的鸡尾酒疗法涉及通过将化学治疗剂与免疫佐剂和藻酸盐 (ALG，alginate) 混合来进行局部化学免疫疗法。ICD 诱导化疗剂响应佐剂刺激原位产生肿瘤疫苗。药物佐剂ALG的原位凝胶化使药物能够缓慢释放，从而降低全身毒性。ICI的组合进一步放大免疫反应并抑制肿瘤转移和复发。

【36】图像引导热消融已被开发为治疗实体瘤的有前途的方法。目前，常用的热消融方法包括射频消融（RFA）、激光消融（LA）、微波消融（MWA）和高强度聚焦超声（HIFU）消融。RFA广泛用于治疗实体瘤，尤其是肝细胞癌（HCC）。RFA 利用射频交流电转化为热量来消融针电极周围的组织并刺激肿瘤特异性 T 细胞反应。 然而，不完全消融和肿瘤复发的问题是 RFA 的缺点【drawbacks缺点】。酪氨酸激酶抑制剂舒尼替尼和 RFA 的组合改善了 HCC 的治疗。RFA导致肿瘤内TSA原位释放，但导致T淋巴细胞PD-1表达上调，这与CD8+ T淋巴细胞耗竭有关。舒尼替尼抑制肝细胞生长因子 (HGF) 抑制肿瘤 T 淋巴细胞中 PD-1 表达的上调。舒尼替尼对 VEGF 的抑制也促进了 DC 活化并抑制了肿瘤血管生成。联合治疗最终导致 CD8+ T 淋巴细胞和 DCs 显着增加以及 Tregs 减少，从而克服了单一治疗的缺点。此外，HIFU作为一种新型的微创消融疗法，已成为癌症治疗的热点。

HIFU 以非侵入性和精确的方式将声能【acoustic energy】传递到目标组织，产生导致肿瘤组织凝固性坏死的高温。HIFU 促进 ICD 和 T 细胞的活化。HIFU 还通过机械破坏间质促进抗原转移到淋巴结和 T 细胞迁移到肿瘤。

【37】ACT 增强效应 T 细胞对癌症的免疫反应，包括肿瘤浸润淋巴细胞 (TIL) 和 CAR-T 细胞。在 TIL 治疗中，肿瘤浸润淋巴细胞从癌症患者中分离出来，在体外扩增，然后再注入患者体内。然而，由于浸润淋巴细胞数量少或 MHC 分子下调，TILs 仅用于治疗少数肿瘤类型，如恶性黑色素瘤。CAR-T细胞涉及对T淋巴细胞进行基因修饰以表达CAR以靶向表达特定抗原的肿瘤细胞。例如，CD19 特异性 CAR-T 细胞已成为治疗 B 细胞恶性肿瘤的金标准。此外，针对白血病和淋巴瘤的CAR-T细胞疗法于2017年获得FDA批准。与 TIL 相比，CAR-T 细胞不受 MHC 限制，可以通过添加共刺激结构域（例如，CD28、OX40 和 4-1BB）进一步增强对肿瘤的免疫反应。该策略利用【 leverages】 CAR-T 细胞对肿瘤抗原的直接识别，并有可能治疗缺乏预先存在的 T 细胞浸润的“冷肿瘤”。表达 IL-7 和 CCL19 的 CAR-T 细胞增加了小鼠实体瘤组织中的 DC 和 T 细胞浸润，并显示出有效的抗肿瘤作用。此外，使用分泌 FLT3L 的 CAR-T 细胞和免疫佐剂导致了类似的结果并诱导了宿主 T 细胞抗原表位扩散。然而，由于肿瘤抗原异质性和对肿瘤的浸润不足，CAR-T细胞在实体瘤中的临床疗效有限。此外，免疫抑制微环境限制了 CAR-T 细胞的肿瘤杀伤作用，因此有必要将 CAR-T 细胞与 ICI 治疗相结合。

【38】治疗性疫苗如肽和肿瘤细胞疫苗、编码新表位的核苷酸疫苗和树突状细胞疫苗一直是令人振奋的临床进展【have been encouraging clinical advances. 】。治疗性疫苗扩大了肿瘤特异性 T 细胞库，增加了 T 淋巴细胞向肿瘤区域的转运，并已成为免疫治疗的新兴方式。Sipuleucel-T 是 FDA 许可的第一种治疗性癌症疫苗，已用于治疗去势抵抗性前列腺癌。Sipuleucel-T 由前列腺酸性磷酸酶 (PAP) 和 GM-CSF 以及自体 DC 构建的融合蛋白 PA2024 组成，可增强抗肿瘤作用。肿瘤新抗原具有高度的肿瘤特异性和免疫原性。在晚期恶性黑色素瘤、非小细胞肺癌和膀胱癌患者的 Ib 临床试验中，个性化新抗原疫苗 NEO-PV-01 与 nivolumab 的组合显着延长了无进展生存期。还观察到新抗原特异性 T 细胞反应、T 细胞运输到肿瘤和诱导表位扩散。mRNA个性化癌症疫苗RO7198457和atezolizumab的组合在晚期实体恶性肿瘤患者的Ib期临床试验中显示出临床益处（NCT03289962）。该疫苗在 77% 的患者中诱导了新抗原特异性 T 细胞反应。这一结果表明，使用个性化癌症疫苗结合免疫检查点阻断可以在患者体内产生特定的免疫反应。然而，操作复杂、流程繁琐、价格高昂，仍然是限制个体化癌症疫苗在癌症治疗中广泛应用的因素。

【39】使用致癌途径抑制剂有助于逆转肿瘤固有的 T 细胞排斥。PAK4 在“冷肿瘤”中大量表达，在 WNT/β-catenin 通路中起关键作用。在小鼠肿瘤模型中敲除 PAK4 或应用 PAK4 抑制剂 KPT-9274 可增强 CTL 在肿瘤中的浸润并提高 PD-1 阻断剂的治疗效果。然而，针对 WNT 的治疗效果仍然存在争议。例如，WNT 通路的内源性抑制剂，例如 dickkopf (DKK) 家族的一些蛋白质，在促进肿瘤免疫逃逸中发挥作用，并与某些癌症的较差预后相关。DKK2 通过与 WNT 通路的细胞表面受体 LRP5 和 LRP6 结合来抑制 WNT-β-catenin 信号传导。DKK2 表达在人类结直肠癌 (CRC) 中上调，并通过抑制 NK 细胞和 CD8+ T 细胞的活化来促进肿瘤进展。这挑战了抑制 WNT 途径将改善免疫治疗的观点。此外，最近的研究表明，内皮细胞中 WNT 通路的激活可促进 T 细胞浸润到肿瘤中并增强 ACT 等免疫疗法的有效性，这表明仍需要进一步研究使用 WNT 抑制剂作为免疫疗法的可行性。事实上，使用 WNT/βcatenin 通路抑制剂的临床数据并不完全支持其在临床上促进免疫治疗的推定功能【putative function】。

【40】PI3K-AKT 通路的激活与 CTL 浸润和功能的抑制有关。PI3Kβ 抑制剂抑制 PTEN 缺陷黑色素瘤细胞系中 AKT 通路的激活并增强 T 细胞介导的杀伤。PI3Kβ 抑制剂和 ICI 的组合显着增加了小鼠肿瘤模型中浸润性 T 淋巴细胞的数量。

【41】RAS 在过去被认为是“不可治疗的”靶点。针对 RAS 癌基因的单一疗法由于多种机制而面临有限的疗效，例如 RAS 下游通路的反馈再激活。然而，最近的研究发现，ARS-1620，一种专门针对 KRAS-G12C 突变体的小分子抑制剂，可显着抑制 KRAS-G12C 肿瘤的生长。该证据表明抑制突变RAS的新治疗途径。此外，MEK 抑制剂与 ICI 联用导致肿瘤浸润性 T 细胞增加和 MDSC 百分比降低，进而显着抑制 TP53/KRAS 驱动的肺癌小鼠模型中的肿瘤生长。BRAF 的致癌突变激活了 RAF-MEK-ERK (MAPK) 通路。抑制 BRAF 或 MEK 可抑制抑制性细胞因子（如 IL-6、IL-10 和 VEGF）的产生或增强黑素细胞分化抗原的表达，从而促进 T 细胞识别黑素瘤。三种靶向 MEK 的变异激酶抑制剂 cobimetinib、trametinib 和 binimetinib 在临床上被批准用于治疗 BRAF V600 突变黑色素瘤。

【42】CDk4/6可与cyclin D结合，使细胞通过RB-EF2通路进入S期，促进肿瘤细胞增殖。用 CDK4/6 抑制剂 abemaciclib 治疗 CT26 同基因小鼠肿瘤导致肿瘤浸润 T 淋巴细胞增加，T 细胞活性显着上调，这可以通过 T 淋巴细胞活化标志物来证明。Abemaciclib 还导致抗原呈递增强，并在与抗 PD-1 治疗一起应用时具有协同作用。

【43】表观遗传疗法可以通过多种机制将肿瘤从免疫“冷”状态转变为免疫“热”状态。表观遗传药物可增强多种趋化因子的表达，如 CXCL9、CXCXL10 和 CCL5，并促进 T 细胞向肿瘤转运。表观遗传疗法还可以诱导 ERVs 并抑制 MYC 信号传导，从而增强 I 型 IFNs 和相关趋化因子的表达。此外，表观遗传疗法可以通过增加肿瘤抗原（如 CTA）的表达和恢复 MHC-I 抗原加工和呈递机制来增强肿瘤免疫原性。DNMT 抑制剂 guadecitabine 上调乳腺肿瘤细胞中 MHC-I 的表达，增强 IFN-γ 反应，并促进 T 细胞向肿瘤募集。此外，guadecitabine 与抗 PD-L1 抗体具有协同作用。这一结果表明将表观遗传抑制剂与 ICI 策略相结合以用于未来临床应用的可行性。多种表观遗传药物已获得 FDA 批准，例如阿扎胞苷【azacitidine】和地西他滨【decitabine】（DNMT 抑制剂）、他泽美司他【tazemetostat】（一种 EZH2 抑制剂）以及恩替司他【entinostat】和伏立诺他【vorinostat 】（HDAC 抑制剂）。

【44】由促血管生成信号和抗血管生成信号【pro-and antiangiogenic signals】之间的失调平衡引起的持续性血管生成【Persistent angiogenesis】是肿瘤的标志之一。抗血管生成疗法 (AT) 在结构和功能上克服了肿瘤血管异常，改善了组织灌注，并增加了免疫效应细胞的浸润。AT 介导的免疫重编程【reprogramming】反过来会改善血管正常化，从而产生增强的正反馈循环。贝伐单抗是 FDA 批准的第一个血管生成抑制剂。转移性肾癌患者在贝伐单抗和阿特珠单抗联合治疗后观察到肿瘤特异性 T 淋巴细胞浸润增加。此外，联合治疗导致血管特征基因【vascular signature genes】（例如，ANGPT2 和 CD31）的表达下调和 CD8+ T 效应基因（例如，CD8A、GZMB 和 IFNG）和 MHC-I 以及趋化因子（例如，分形碱）的上调。这些结果表明肿瘤特异性 T 细胞浸润的增加可能是由于联合治疗介导的淋巴细胞运输增强。此外，在针对不可切除肝细胞癌患者的 III 期 IMbrave150 试验中，与索拉非尼治疗的结果相比，atezolizumab 联合贝伐单抗治疗显着改善了总生存期和无进展生存期结局。鉴于血管正常化和免疫重编程之间的关系，联合治疗有望进一步逆转免疫抑制微环境，提高免疫治疗的疗效。

【45】考虑到 TGFβ 的免疫抑制功能，基于 TGFβ 抑制的治疗已被证实是促进 T 淋巴细胞浸润的有效方法。TGFβ 与非炎症 T 细胞表型缺乏免疫反应有关。在具有免疫排斥表型【immune-excluded phenotype】的乳腺癌小鼠模型中，抗 PD-L1 和抗 TGFβ 抗体的联合治疗显着降低了肿瘤负荷并增加了肿瘤浸润性 T 细胞，尤其是 CD8+ T 效应细胞。Galunisertib 是一种抑制 TGFBR1 激酶活性的小分子，是测试最广泛的化合物。Galunisertib 治疗增加了小鼠结肠直肠模型中的 T 细胞浸润并提高了对检查点治疗的敏感性。TGFβ阻碍【impedes】放射治疗后原位肿瘤疫苗的产生。用阻断全身性 TGFβ 活性的 1D11 抗体治疗可增强 T 细胞在皮下肿瘤照射后对内源性肿瘤抗原的反应。

【46】CXCR4 是 CXCL12 的受体，在多种肿瘤中过度表达。CXCL12-CXCR4 轴在从肿瘤区域隔离【sequestration[ˌsiːkwəˈstreɪʃn]封存;扣押;隔离;没收;螯合作用】 CTL 以减少 CTL 浸润和介导免疫抑制细胞浸润到肿瘤中起间接作用。在 PDAC 模型中，用 CXCR4 抑制剂 AMD3100 抑制 CAF 介导的 CXCL12/CXCR4 轴促进了 T 细胞积累和癌症消退。此前的研究表明，派姆单抗等免疫疗法对胰腺癌等“冷肿瘤”无效。然而，在 COMBAT 试验中，转移性 PDAC 与 CXCR4 拮抗剂 BL8040 和派姆单抗的协同治疗【synergistic treatment】增加了肿瘤浸润性 CD8+ 效应 T 淋巴细胞，降低了肿瘤中 MDSC 的密度，并减少了循环 Treg 的数量。这些结果表明，调节某些趋化因子有助于肿瘤特异性 T 淋巴细胞归巢到肿瘤并逆转免疫抗性。

【47】多种纳米药物与免疫疗法相结合，有助于“冷肿瘤”向“热肿瘤”的转变（图5）。纳米药物具有三种不同的靶向途径：肿瘤细胞、TME 和外周免疫系统。基于纳米药物的靶向肿瘤治疗包括被动靶向和主动靶向。被动靶向【Passive targeting】可以通过EPR效应促进纳米药物在肿瘤中的选择性积累。然而，最近的研究表明，纳米材料的被动靶向能力可能与胞吞作用【transcytosis】有关。主动靶向涉及使用特异性识别肿瘤细胞表达的特定受体的靶向配体（例如肽、抗体和转铁蛋白）。

用纳米药物改善 T 细胞浸润。纳米药物具有三种不同的靶向途径：肿瘤细胞、TME 和外周免疫系统。 (A) 包括光热疗法 (PTT，photothermal therapy)、光动力疗法 (PDT，photodynamic therapy)、磁热疗 (MH，magnetic hyperthermia) 和高强度聚焦超声 (HIFU，high-intensity focused ultrasound ) 在内的多种方法可以通过促进肿瘤抗原和损伤相关分子模式 (DAMP，damage-associated molecular patterns) 的释放来诱导 ICD。释放的 DAMP 作为佐剂增强肿瘤的免疫原性，并与释放的肿瘤抗原一起促进树突状细胞 (DC) 活化和 T 细胞启动。(B) 当靶向 TME 时，纳米药物抑制免疫抑制细胞和免疫抑制分子（例如，TGFβ）并增强 T 细胞的活性。(C) 当外周免疫系统被靶向时，纳米药物被设计用于增强肿瘤抗原呈递和 T 细胞在淋巴结中的启动。

Adapted with permission from 150, copyright 2019 American Chemical Society.

【48】靶向肿瘤细胞的纳米药物可以诱导 ICD 并增强肿瘤免疫周期 。例如，doxil 是化疗药物阿霉素的聚乙二醇化脂质体，通过 ICD 促进 DC 和 CD8+ T 细胞增殖并抑制 Treg 浸润。Doxil 还与 ICI（抗 PD-1 和抗 CTLA-4）具有协同作用，并显示出比游离多柔比星更高的疗效。当靶向 TME 时，纳米药物抑制免疫抑制细胞（例如，M2 TAM 和 MDSC）和免疫抑制分子（例如，TGFβ 和 ADO），它们还可以增强效应免疫细胞（例如，巨噬细胞和 CTL）的活性和功能。将 TGFβ 受体抑制剂 SB525334 加载到靶向 ACT T 细胞的脂质体上。这种纳米药物在黑色素瘤小鼠中抑制 TGFβ 表达并促进 T 细胞活化以及肿瘤消退。用 IL-2 和激动性抗 4-1BB 加载脂质体可增强 CTL 的肿瘤浸润以及细胞因子的产生和颗粒酶的表达。当靶向外周免疫系统时，纳米药物旨在促进 DLN 中的抗原呈递和 CTL 产生。基于纳米药物的疫苗改善了向淋巴结的抗原递送，促进了抗原交叉呈递，并提高了 CTL 激活水平。此外，纳米药物已被设计为通过替换次级淋巴结中的 APC 直接促进 T 细胞启动。使用仿生磁小体【biomimetic magnetosomes】作为人工 APC 的特点是磁性纳米簇被白细胞膜包裹并被修饰以刺激膜上的信号。人工APCs不仅能扩增和刺激CTLs，还能有效引导回输的CTLs进入肿瘤组织，从而抑制肿瘤生长。

肿瘤光疗 (PT)

【49】PT，包括光热疗法（PTT，photothermal therapy）和光动力疗法（PDT，photodynamic therapy），已被开发为实体瘤，尤其是浅表肿瘤的潜在治疗方法。PTT使用具有光热转换能力的光热剂（如有机纳米颗粒、金纳米颗粒和氧化石墨烯）吸收近红外（NIR）激光并将其转化为热能来杀死肿瘤细胞。PDT 涉及使用激光照射已递送光敏剂的肿瘤，以激活光敏剂并产生细胞毒性 ROS。它可以引起细胞核中的DNA损伤，从而诱导肿瘤细胞死亡。与其他消融方式（例如射频消融和微波消融）相比，由于光敏剂在肿瘤中的积累和光的可控性，PT 更具选择性，对周围组织的毒性更小。

【50】PTT和PDT分别通过热损伤和化学损伤诱导ICD，增强CTL的浸润和免疫治疗反应。基于逐层【layer-by-layer】Apt/PDGs^s@pMOF纳米平台的PDT增强了小鼠三阴性乳腺癌的免疫原性，选择性抑制了MDSCs，促进了向“热肿瘤”的转变。然而，由于光穿透有限、PTT 引起的热休克反应以及 PDT 对氧气的依赖性，单次 PT 治疗显示出有限的功效。PDT和PTT的结合可以实现有希望的协同抗肿瘤作用。混合纳米卟啉 (Pp18-Lips) 介导的协同 PTT/PDT 导致肿瘤浸润性 CTL 和炎性细胞因子（例如，TNF-α 和 IFN-γ）增加和 Tregs 减少。协同 PDT/PTT 比单独使用 PDT 或 PTT 产生更强的抗肿瘤免疫反应和更强的肿瘤抑制 。PT 还与免疫疗法协同，因为 PT 通过介导 ICD 增强肿瘤的免疫原性，而免疫疗法增强了肿瘤的“异位效应”治疗。PTT 介导的 GOP@aPD-1 纳米颗粒有效地将抗 PD-1 递送至黑色素瘤细胞，并将 ICI 治疗与肿瘤靶向 PTT 相结合。这种组合导致 CD8+ T 细胞浸润增加，PD-1 阻断在小鼠黑色素瘤中的功效提高，并抑制了肿瘤生长。

【51】MH是指在交变磁场（AMF）存在的情况下，通过磁性纳米粒子（MNP）的滞后和弛豫效应，将磁能转化为热能，从而选择性加热肿瘤。MH除了可以加热杀死肿瘤细胞外，还可以诱导ICD，在癌症治疗中显示出巨大的潜力。与 PT 和热消融疗法相比，MH 没有穿透深度限制，可以更有效地靶向和更精确地控制加热温度。氧化铁纳米药物ferumoxytol促进巨噬细胞极化为促炎M1表型。如 caspase-3 裂解增加所示，该开关【 switch 】诱导了肿瘤细胞的凋亡。另一个典型的例子是亚铁磁涡旋域氧化铁纳米环（FVIOs，ferrimagnetic vortex-domain iron oxide nanorings ），它具有优异的纳米磁性。FVIO介导的轻度MH诱导4T1乳腺肿瘤细胞表面CRT表达并促进T细胞活化。FVIO 导致 CTL 浸润显着增加，同时 MDSC 减少，并且与抗 PD-L1 抗体具有协同作用。

【52】HIFU与免疫疗法相结合取得了显着的治疗效果。与单独的免疫疗法或单独的 HIFU 相比，HIFU 和 TLR 激动剂 CpG 在小鼠黑色素瘤中的组合增强了 DLN 中的抗原交叉呈递、I 型 IFN 的释放以及与 T 细胞启动和刺激相关的基因（例如，Eomes , Prf1 和 Icos)。然而，HIFU 的研究仍然不足，缺乏研究证明 HIFU 单一疗法可以控制肿瘤。该领域的现状表明，HIFU 可以促进 T 细胞启动和肿瘤消退，但可能需要诱导额外的免疫佐剂。HIFU 杀死肿瘤细胞也与空化效应【cavitation effects】有关，空化效应可以有效地聚集能量。最近的一项研究表明，将微泡【microbubbles 】和编码 IFN-β 的质粒 DNA 直接注射到小鼠模型中的肿瘤中，然后应用低频超声 (250 kHz) 分解肿瘤，可以去除大量肿瘤细胞和同时实现CTL的大规模渗透。剩余的肿瘤细胞也形成了膜孔，允许细胞的基因转染并触发抗肿瘤免疫反应。

【53】考虑到肿瘤部位 T 淋巴细胞浸润与 ICI 治疗预后的相关性，确定 T 细胞反应缺失的原因至关重要。在这篇综述中，我们总结了抑制 T 细胞浸润的各种机制，例如肿瘤抗原加工和呈递过程中的缺陷、内源性致癌途径激活、异常脉管系统和趋化因子以及 TME 抑制。其他影响 T 细胞浸润的已知和未知因素，如 TLS 和微生物组，仍有待评估。TLSs 具有淋巴结样功能，与 T 细胞浸润肿瘤和对免疫治疗的良好反应有关。探索增强 TLS 形成和功能的治疗策略可能会促进靠近肿瘤的 DC 激活幼稚 T 淋巴细胞并改善对癌症免疫治疗的反应。与“冷肿瘤”相比，“热肿瘤”对 ICI 单药治疗的反应更佳。因此，通过干预措施促进“冷肿瘤”向“热肿瘤”转变，有助于降低对 ICI 的耐药性。此外，我们随后讨论了各种改善 T 细胞浸润的治疗措施，例如致癌途径抑制剂、抗血管治疗、ACT、疫苗、溶瘤病毒和细胞毒治疗。ICI 与这些疗法的结合可逆转 T 细胞耗竭，增强疗法的“异位效应”，并展示增加的临床疗效。然而，联合治疗的最佳剂量和给药顺序需要进一步评估，以优化 T 细胞功能、促进 T 细胞记忆并避免过度激活。此外，还有一些问题需要解决，例如药物的非特异性分布，以及治疗引起的全身性不良反应。

【54】随着纳米技术的发展，纳米药物和生物材料辅助的局部治疗为未来提供了新的机遇。纳米药物的使用提高了药物的精确度和生物利用度，减少了免疫疗法引起的副作用，并通过 EPR 效应和主动靶向使药物在肿瘤中选择性积累。如上所述，PTT、PDT、MH和HIFU都能够诱导从头【de novo】抗肿瘤反应，这是通过诱导ICD来实现的。这些方法不需要事先了解肿瘤抗原并诱导内源性个性化原位疫苗的产生。然而，通过非炎症性细胞凋亡或消融杀死实体瘤并不能使肿瘤细胞具有足够的免疫原性。与细胞凋亡相比，细胞焦亡【pyroptosis 】是一种促炎形式的细胞死亡，会导致大量炎症分子（例如，IL-1β 和 IL-18）的释放，从而激发强大的抗肿瘤 T 细胞反应（图 6）。使用纳米技术诱导细胞焦亡增加了肿瘤细胞的免疫原性，并可能有效改善肿瘤中的 T 细胞浸润 。例如，Zhao 等人设计了负载吲哚菁绿 (ICG) 和地西他滨的仿生纳米颗粒 (BNP)，用于光诱导细胞焦亡。由于 GSDME 基因的启动子甲基化，GSDME 在大多数肿瘤细胞中的表达比在正常细胞中低得多。作为 DNA 甲基化抑制剂，地西他滨通过上调 GSDME 表达促进 caspase-3 裂解为 GSDME，从而导致肿瘤细胞焦亡。由 BNP 介导的细胞焦亡导致肿瘤细胞释放大量炎症分子，并诱导 DLN 中的 DC 成熟和 T 细胞活化，显示出对原发性和远处肿瘤的强大免疫反应。

Schematic illustration of pyroptosis and size-transformable nanoparticles. 焦亡和尺寸可转换纳米粒子的示意图。(A) 多种纳米药物调节介导细胞焦亡过程的半胱天冬酶蛋白的表达。活化的半胱天冬酶将 gasdermin (GSDM) 切割成两个片段：C 端结构域和 N 端结构域。裂解后，gasdermin-N 结构域导致细胞膨胀并出现大气泡。Gasdermin 诱导的细胞焦亡导致大量促炎分子的释放和 T 细胞的活化。(B) 使用可改变尺寸的纳米粒子来延长循环时间并实现深度渗透。

【55】然而，必须克服纳米药物使用的某些挑战，例如血液循环时间短，以及在肿瘤组织中的渗透和积累不足。在光疗或化疗中使用可改变尺寸的纳米粒子可以实现纳米药物的深度渗透并改善 T 细胞对肿瘤的浸润。粒径较大的纳米载体利用EPR效应延长纳米药物的循环时间，改善其在肿瘤组织中的积累。到达肿瘤部位后，纳米药物会响应 pH 值或酶发生尺寸变化，并释放转化的小纳米颗粒，这些纳米颗粒表现出有效的肿瘤组织渗透，因此可以被肿瘤细胞有效内化。

【56】此外，小分子纳米抗体在影像诊断中的应用，可以更方便、更全面地评估TME中T细胞浸润的程度，为实现肿瘤诊疗一体化提供新思路。更好地了解这些方面将有利于指导个性化癌症免疫治疗，并将 ICI 治疗的益处扩展到更广泛的患者群体。

原文：

【1】Immunotherapy, represented by immune checkpoint inhibitors (ICIs), has greatly improved the clinical efficacy of malignant tumor therapy. ICI-mediated antitumor responses depend on the infiltration of T cells capable of recognizing and killing tumor cells. ICIs are not effective in "cold tumors", which are characterized by the lack of T-cell infiltration. To realize the full potential of immunotherapy and solve this obstacle, it is essential to understand the drivers of T-cell infiltration into tumors. We present a critical review of our understanding of the mechanisms underlying “cold tumors”, including impaired T-cell priming and deficient T-cell homing to tumor beds. “Hot tumors” with significant T-cell infiltration are associated with better ICI efficacy. In this review, we summarize multiple strategies that promote the transformation of "cold tumors" into “hot tumors” and discuss the mechanisms by which these strategies lead to increased T-cell infiltration. Finally, we discuss the application of nanomaterials to tumor immunotherapy and provide an outlook on the future of this emerging field. The combination of nanomedicines and immunotherapy enhances cross-presentation of tumor antigens and promotes T-cell priming and infiltration. A deeper understanding of these mechanisms opens new possibilities for the development of multiple T cell-based combination therapies to improve ICI effectiveness.

【2】Recently, immune checkpoint inhibitors (ICIs), such as nivolumab and pembrolizumab, have been applied to an increasing number of cancer types, forming a paradigm treatment in clinical trials 1, 2. Although ICIs have shown clinical activity in a wide range of tumor types, a substantial percentage of patients still do not respond to ICI therapy 3. ICI-mediated antitumor responses rely on the expression of PD-L1 in tumors and the infiltration of T cells capable of recognizing and killing tumor cells. Immune cells such as CD8+ T cells are associated with prolonged survival of cancer patients and increased efficacy of immunotherapy 4. A lack of T cells in tumors can lead to resistance to immunotherapy 5. The success of chimeric antigen receptor (CAR) T-cell infusions for patients' leukemia and lymphoma also demonstrates the importance of T cells in antitumor immunity 6. Considering the potential mechanisms of cancer immunotherapy, the infiltration of CD8+ T lymphocytes in tumors is important for the therapeutic response to ICIs.

【3】According to the spatial distribution of cytotoxic immune cells in the tumor microenvironment (TME), a tumor is classified into one of three basic immunophenotypes: immune-inflamed, immune-excluded and immune-desert phenotypes (Figure 1) 3. Immune-inflamed tumors, also named “hot tumors”, are characterized by high T-cell infiltration, increased interferon-γ (IFN-γ) signaling, expression of PD-L1 and high tumor mutational burden (TMB) 7. Tumors with an inflamed phenotype tend to be more responsive to ICIs 8, 9. Immune-excluded tumors and immune-desert tumors can be described as “cold tumors”. In immune-excluded tumors, CD8+ T lymphocytes localize only at invasion margins and do not efficiently infiltrate the tumor 10. In immune-desert tumors, CD8+ T lymphocytes are absent from the tumor and its periphery 10. In addition to poor T-cell infiltration, “cold tumors” are characterized by low mutational load, low major histocompatibility complex (MHC) class I expression and low PD-L1 expression 7. Immunosuppressive cell populations, including tumor-associated macrophages (TAMs) and T-regulatory cells (Tregs) and myeloid-derived suppressor cells (MDSCs), are also present in cold tumors 7. These features suggest that cold tumors lack innate immunity or that the innate antitumor immune features present in “cold tumors” may be ineffective due to the exclusion of immune cells 3. In contrast to the inflamed phenotype, cold tumors rarely respond to ICI monotherapy 9.

Tumor immune phenotypes. Based on the spatial distribution of CD8+ T lymphocytes in the tumor microenvironment (TME), a gradient of three immunophenotypes is observed: the immune-desert, immune-excluded and immune-inflamed phenotypes. In the immune-desert phenotype, immune cells are absent from the tumor and its periphery. In the immune-excluded phenotype, immune cells accumulate but do not efficiently infiltrate. In the immune-inflamed phenotype, immune cells infiltrate but their effects are inhibited. Notably, the three different phenotypes have different response rates to immune checkpoint inhibitors.

【4】Driving T cells into the TME is a gradual process (Figure 2): tumor cell death and antigen release, antigen-presenting cell (APC) processing and presentation of tumor antigens, and APC and T-cell interactions lead to T-cell priming and activation 11. Ideally, once activated, these tumor-specific T cells exit lymph nodes and travel through the bloodstream to tumor site 11. The production of T cells and their physical contact with tumor cells is crucial for the success of antitumor immunity 12. Once infiltrating the tumor bed, cytotoxic T lymphocytes (CTLs) specifically recognize antigenic peptide-MHC complexes on the surface of tumor cells, form immune synapses, and release perforin and granzyme to destroy the tumor cells 13. In addition, CTLs contribute to the apoptosis of tumor cells through the Fas/FasL pathway and suppress tumors by inducing ferroptosis and pyroptosis 14. Dead tumor cells release additional tumor antigens and thereby amplify the T-cell response 11.

The tumor-immunity cycle and three immunophenotypes. Antitumor immunity is mediated to a large extent by CD8+ T lymphocytes. The tumor-immunity cycle consists of the following steps: (1) tumor antigen release, (2) tumor antigen processing and presentation, (3) T-cell priming and activation, (4) trafficking of T lymphocytes through the bloodstream to tumors, (5) infiltration of T lymphocytes into the tumor parenchyma from the vasculature or tumor periphery, (6) recognition of tumor cells, and (7) cytotoxic T lymphocyte (CTL) destruction of tumor cells by granule exocytosis or through the Fas/FasL pathway. Dead tumor cells release additional antigens, allowing the tumor-immunity cycle to continue. Notably, tumors with the immune-desert phenotype (yellow) cannot pass steps 1-3 due to the absence of T lymphocytes in both the tumor and its margins. Tumors with the immune-excluded phenotype (blue) cannot exceed steps 4-5 due to a lack of T lymphocytes in the tumor bed. Tumors with the immune-inflamed phenotype (red) cannot exceed steps 6-7 due to T-cell exhaustion and checkpoint activation. Adapted with permission from 11, copyright 2013 Elsevier.

【5】With the development of nanotechnology, immunotherapy based on nanomedicines and biomaterials offers new opportunities for the future. Nanomedicines offer unique advantages in oncology treatment, such as improved drug precision and bioavailability and reduced immunotherapy-induced side effects 15. In addition, nanomedicines promote selective accumulation in tumors through enhanced permeability and retention (EPR) effects or include high-affinity ligands to achieve active targeting of tumors 15. Nanotechnology-based immunotherapy enhances tumor-specific immune responses, promotes the infiltration of CTLs, and inhibits tumor metastasis and recurrence.

【6】Given the importance of T-cell infiltration, understanding the mechanisms of T-cell homing to the tumor is necessary. To improve the clinical benefit of immunotherapy, ICIs may be combined with strategies that convert “cold tumors” to “hot tumors”, which may make these tumors more sensitive to ICI therapy. In this review, we summarize the various mechanisms of T-cell infiltration disorders and current approaches to directing T cells into tumors. Finally, we summarize recent advances, challenges and opportunities for nanomedicine-based local therapeutic strategies to enhance T-cell infiltration and discuss further prospects in this field.

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【7】ICI response rates are low in “cold tumors”, as characterized by the absence of T-cell infiltration. In the process of driving T cells into tumors, there are many factors that can influence T-cell priming and T-cell homing to the tumor bed, leading to a noninflamed T-cell phenotype and failed antitumor immunity (Figure 3).

Mechanisms of three distinct tumor phenotypes. Three different phenotypes are associated with specific biological mechanisms. Tumors with the immune-desert phenotype (yellow) may lack T-cell priming due to the absence of tumor antigens, defective antigen processing and presentation machinery, or impaired DC-T-cell interactions. Tumors with the immune-excluded phenotype (blue) may exhibit activation of oncogenic pathways, aberrant chemokines, aberrant vasculature and hypoxia, or an immunosuppressive tumor microenvironment (e.g., stromal barriers). Tumors with the immune-inflamed phenotype (red) can be infiltrated by many immune cells, but these immune cells are suppressed due to checkpoint activation. ADO: adenosine; ATP, adenosine triphosphate; B2M: beta-2-microglobulin; BATF3: basic leucine zipper ATF-like transcription factor 3; CAFs: cancer-associated fibroblasts; CRT, calreticulin; CTLA4, cytotoxic T lymphocyte-associated antigen-4; CXCL: CXC-chemokine ligand; DNMT: DNA methyltransferase; ECM: extracellular matrix; ETBR: endothelin B receptor; EZH2: enhancer of zeste homolog 2; FLT3L: Fms-like tyrosine kinase 3 ligand; GM-CSF: granulocyte-macrophage colony-stimulating factor; HDAC: histone deacetylase; HEV: high endothelial venule; HMGB1: high mobility family protein B1; ICAM: intercellular adhesion molecule; IDO: Indoleamine 2,3-dioxygenase; IFN: interferon; IL: interleukin; MDSC: myeloid-derived suppressor cell; MHC: major histocompatibility complex; PD-1, programmed cell death protein 1; PD-L1, PD-1 ligand; STC1: stanniocalcin 1; TAM: tumor-associated macrophage; TAP: transporter associated with antigen processing; TGFβ: transforming growth factor-β; TIM3, T cell immunoglobulin and mucin domain-containing 3; TLR: Toll‑like receptor; TLS: tertiary lymphoid structure; TME: tumor microenvironment; Treg: T-regulatory cell; VCAM: vascular cell adhesion molecule; VEGF: vascular endothelial growth factor.

【8】The most direct cause of T-cell priming disorders is insufficient T-cell recognition due to a lack of tumor antigens. Table 1 summarizes the mechanisms of defects in T-cell priming. In general, targeted tumor antigens can be classified into two broad categories: nonmutated self-antigens and neoantigens generated by nonsynonymous somatic mutations 16. Self-antigens include nonmutated proteins that are aberrantly expressed or overexpressed in tumor cells, such as tumor-associated antigens (TAAs) and cancer/testis antigens (CTAs). Although self-antigens also elicit a tumor immune response, the primary target of the immune response is neoantigens, also termed tumor-specific antigens (TSAs). Neoantigens are specific to tumor cells and arise from somatic mutations in cancer genomes 16. The recognition of tumor neoantigens may promote T-cell priming and infiltration and can lead to a long-term clinical response 11, 16.

【导读

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【9】The TMB is broadly characterized as the number of total nonsynonymous single-nucleotide mutations in a tumor. In general, tumors with a higher TMB are believed to carry a higher neoantigen load that can be recognized by T cells, making them more likely to prime the immune system 17. Significant associations between high TMB and improved response to ICIs have been reported in a variety of tumor types 18, 19. The TMB has been used as a novel biomarker to predict the efficacy of programmed cell death protein 1 (PD-1) inhibitors 17, 19. Consistent with the importance of the efficacy of ICIs, a high TMB was associated with greater immune cell infiltration 20. Furthermore, a mutliomics network analysis revealed that in tumors characterized by recurrent mutations, such as melanoma and colorectal cancer, mutation or neoantigen burden was positively correlated with CTL infiltration 21. Considering the relationship between high TMB and tumor-specific T cells, low mutational load or neoantigen load contributes, in part, to the lack of immune infiltration and the acquisition of ICI resistance. However, in tumors characterized by recurrent copy number alterations, such as breast cancer, a correlation between tumor-specific T-cell infiltration and neoantigen load is lacking 21. An investigation of data on 266 melanomas in The Cancer Genome Atlas (TCGA) revealed no difference in antigen expression between cold and hot tumors 22. This finding suggests that other mechanisms, in addition to those involving low TMB, contribute to the absence of T-cell infiltration.

【10】After recognizing tumor antigens, APCs process the antigens and express the corresponding antigen peptide-MHC class I complex on its surface. However, alterations in the APM, such as downregulation of MHC-I molecule expression or the absence of beta-2-microglobulin (B2M), limit the presentation of antigen peptide-MHC class I complexes in the presence of tumor antigens. During antigen processing and presentation, transporters associated with antigen processing (TAP) transport cytosolic cleaved antigens to the endoplasmic reticulum for binding to the MHC. The deletion in TAP is related to defects in the antigen presentation process, which further affects the priming of T lymphocytes 23. B2M, the invariant chain of the MHC, is critical for the successful folding and transport of MHC-I to the cell surface 5. Knocking down the B2M gene in the M202 and M233 human melanoma cell lines resulted in the absence of MHC-I molecules expressed on their surface, and the absence of tumor-specific T-cell recognition and cytotoxicity 24. Identical results were observed in a B2M-knockout mouse model of lung cancer that showed resistance to PD-1 blockade 25. In addition, the lysosomal pathway has been linked to the reduced infiltration of CD8+ T lymphocytes. In pancreatic ductal adenocarcinoma (PDAC), the autophagy-associated receptor NBR1 induces the degradation of MHC-I on the cell surface of tumor cells, which in turn affects T-cell responses 26. These findings suggest that defects in tumor antigen processing and presentation pathways inhibit T-cell priming and the effectiveness of cancer immunotherapies.

【11】Dendritic cells (DCs) are professional APCs with the unique ability to acquire antigens, migrate to secondary lymphoid organs (e.g., lymph nodes and spleen), and initiate the in vivo immune response. DC activation requires that pattern recognition receptors (PRRs) on their surface recognize “danger signals”, including pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) 27. This recognition enables DCs to present a tumor antigen peptide-MHC class I complex to T cells upon contact with them. DCs also express costimulatory signals such as B7 (including CD80 and CD86), providing the secondary signaling necessary for T-cell activation 28. Tumor cells can mediate diminished phagocytosis of DCs by trapping “danger signals”. For example, stanniocalcin 1 (STC1), an intracellular checkpoint, can trap DAMPs (e.g., calreticulin (CRT)) and inhibit DC phagocytosis and T-cell activation, contributing to tumor immune escape. Furthermore, STC1 is associated with low T-cell activation and poor survival in melanoma patients 29.

【12】DCs are generally classified into two broad categories: plasmacytoid DCs (pDCs) characterized by the production of IFN-α, and conventional DCs (cDCs), which effectively stimulate T-cell proliferation 30. CDCs are further categorized into two distinct subsets: BATF3-dependent DCs and IRF4-dependent DCs 30. BATF3 DCs have the ability to cross-present tumor-derived antigens through the MHC-I pathway and thus initiate T cells 31. Furthermore, BATF3 DCs are the primary source of CXC-chemokine ligand 9 (CXCL9) and CXCL10, two key chemokines required to recruit CD8+ T cells expressing CXCR3 to tumors. There is a significant correlation between BATF3 DC markers (e.g., BATF3 and IRF8), the expression of CXCL9, CXCL10, and CXCL11 and the CD8+ effector T-cell phenotype in melanoma 22, 32. In the absence of BATF3 DCs, CD8+ effector T cells fail to migrate to tumors and antitumor immunity is thus defective 32. This finding validates the notion that BATF3 DCs may be essential for the priming and recruitment of the endogenous T cells necessary to counteract tumors.

【13】The regulation of Fms-like tyrosine kinase 3 ligand (FLT3L) and granulocyte-macrophage colony-stimulating factor (GM-CSF) is important for the differentiation and recruitment of DCs 33. FLT3L is a growth factor that promotes the differentiation of hematopoietic progenitor cells from the bone marrow to the DC lineage 33. Tumor-derived FLT3L increased the infiltration of BATF3 DCs and CD8+ T lymphocytes in mouse tumors and enhanced migratory and resident DC subsets in draining lymph nodes (DLNs), suggesting a mobilizing effect of FLT3L on DC cells 34. Deficiency of FLT3L or GM-CSF resulted in a reduced number of DCs in secondary lymphoid organs and attenuated T-cell immune responses 35. Given the important role of DC-T cell crosstalk in naïve T-cell priming, impaired DC activation, a lack of DCs, and the overexpression of cosuppressive signals can lead to impaired T-cell activation.

【14】For the purposes of this review, we summarize the mechanisms by which T cells are prevented from homing to the tumor bed (Table 2). There is growing evidence showing that the activation of tumor cell oncogenic pathways is related to the “cold tumor” phenotype and the potential for immunotherapy resistance. In WNT/βcatenin-positive melanoma tumors, reduced production of CCL4 results in decreased recruitment of BATF3 DCs to the TME 36. Ultimately, in the absence of the CXCL9 and CXCL10 produced by BATF3 DCs, CTLs are not recruited to the tumor 32. An analysis of human metastatic melanoma samples showed a negative correlation between CD8A expression and activation of the β-catenin signaling pathway 36. Direct injection of BATF3 DCs helped restore T-cell infiltration in β-catenin-positive tumors and resulted in modest tumor suppression 36. This outcome suggests that WNT/β-catenin signaling activation and defective BATF3 DC recruitment mediate T-cell exclusion and tumor cell escape from the immune system. Notably, activation of this oncogenic pathway excludes CTLs only when β-catenin is located into the nucleus 37. This finding indicates that the exclusion mechanism of CTLs is related to a transcriptional program specifically induced by β-catenin. In addition, only 48% of “cold” melanomas show active βcatenin signaling, suggesting that other oncogenic pathways may mediate immune exclusion 36.

【15】Loss of PTEN activates the PI3K/AKT pathway, which is related to a noninflamed T-cell phenotype and immune resistance of melanoma. Loss of PTEN expression has been found to reduce the lipidation of the autophagosome protein LC3, resulting in decreased autophagic activity, which inhibits T-cell priming and the T-cell-mediated antitumor response 38. CD8+ T-cell infiltration in PTEN-deficient melanoma was significantly reduced compared to that in PTEN-expressing tumors. The results from a TCGA dataset analysis indicated that the expression of T-cell effector molecules (e.g., IFN-γ and granzyme B) was significantly reduced in melanomas with low PTEN expression 38.

【16】As the gene with the most common mutations associated with cancer progression, RAS can lead to the activation of multiple signaling pathways, such as MAPK and PI3K, driving tumorigenesis 39. In addition, oncogenic K-RAS mutations mediate inflammation and crosstalk with the TME. For example, oncogenic K-RAS mutations induce tumor-promoting inflammation through the production of inhibitory cytokines (e.g., IL-6 and IL-8), the activation of NLRP3 inflammasome, and the release of chemokines (e.g., CCL5 and CCL9) 39.

【17】Furthermore, oncogenic signaling through MYC enhances the expression of CD47 and PD-L1 on tumor cells. CD47 binds to inhibitory receptor signal-regulated protein-α (SIRPα) on the surface of APCs such as macrophages and DCs, which can prevent phagocytosis of tumor cells and interfere with antigen uptake 31, 40. Oncogenic KRAS and MYC synergistically induce immune regulation. For example, co-activation of KRAS and MYC in a mouse lung cancer model leads to the production of CCL9 and IL-23. This mediates stromal reprogramming, promotes angiogenesis, and excludes T and B cells and NK cells from tumors 41. It has also been found that inactivating mutations of LKB1 in non-small cell lung cancer (NSCLC) are related to increased neutrophil and decreased T-cell infiltration in a preclinical mouse model 42. In addition, CDK4/6 and STAT3 activation is associated with a noninflamed T-cell phenotype 43-45. Taken together, these results reveal that the activation of oncogenic pathways can affect not only tumor cells but also T cell-mediated antitumor immunity.

【18】The interaction between some chemokine receptors on effector T lymphocytes and corresponding chemokines may affect the trafficking of effector T lymphocytes to tumor sites. The lack of several chemokines, including CXCL9, CXCL10, CCL4, CCL5, CXCL16 or CX3CL1, has been reported to lead to T-cell exclusion 46, 47. Considering the importance of the TH1-type chemokines CXCL9 and CXCL10 to T-cell recruitment, certain tumors show low levels of CXCL9 and CXCL10 expression, which may explain the reduced infiltration of effector T lymphocytes into these tumor beds 32, 36. For example, BATF3 DCs are the major sources of CXCL9 and CXCL10, and a lack of BATF3 DCs leads to low expression of CXCL9 and CXCL10. In addition, epigenetic regulation in tumors is also important for maintaining low expression levels of these cytokines. DNA methyltransferase (DNMT) and enhancer of zeste homolog 2 (EZH2) can mediate DNA methylation and histone lysine methylation, respectively, to suppress the expression of CXCL9 and CXCL10 in ovarian cancer 48. Similar results have been confirmed in colon cancer 49. In preclinical models, treatment with epigenetic modulators promoted tumor infiltration of effector T cells and enhanced the effect of anti-PD-L1 48. In addition to CXCL9 and CXCL10, CCL5 expression is positively related to CD8+ T-cell infiltration 21, 47. The binding of CCL5 to CCR5 promotes the recruitment of CD8+ T cells. However, DNA methylation leads to deletion of CCL5 expression, which in turn contributes to the absence of CD8+ T-cell infiltration 50. In mouse models of NSCLC, the combined use of DNMT inhibitors and histone deacetylase (HDAC) inhibitors increased the expression of endogenous retrovirus (ERV), which in turn induced type I IFN responses. This combination treatment reversed the immune resistance of NSCLC models by downregulating oncogenic MYC signaling, leading to an increase in CCL5 and promoting T-cell infiltration into tumors 51.

【19】However, some chemokines are detrimental to the trafficking of T cells to tumor beds. Stromal cells, especially cancer-associated fibroblasts (CAFs), are the main producers of CXCL12. CXCL12 produced by CAFs misdirects CTLs to the extratumoral stroma and prevents CTLs from entering the tumor 52. Furthermore, elevated CXCL8 expression has been reported to be associated with a reduction in the number of T cells in tumors, increased neutrophil and monocyte infiltration, and limited responses to ICIs 53, 54. These results reveal the regulatory effect of chemokine receptor and ligand interactions on CTL homing to tumors and their integration into the TME.

【20】Adequate T-cell infiltration in the tumor bed is not only dependent on the recruitment of the appropriate chemokines but is also controlled by the tumor vasculature. During the trafficking of CD8+ T lymphocytes to a tumor, they must enter the tumor circulatory system, adhere to vascular endothelial cells and migrate across the vessel wall 11. The recruitment of CD8+ T cells to tumors requires the action of vascular endothelial adhesion molecules, including P-and E-selectin, intercellular adhesion molecules (ICAMs), and vascular cell adhesion molecules (VCAMs) 55, 56. However, the downregulation or ineffective aggregation of adhesion molecules on tumor endothelial cells leads to endothelial cell anergy and reduced effector T-cell trafficking to tumor sites 55, 57. Endothelin binds to a corresponding receptor on endothelial cells, endothelin B receptor (ETBR), and reduces ICAM-1 production, thereby inhibiting CD8+ T cell adhesion to endothelial cells 58. Additionally, vascular endothelial growth factor (VEGF), produced by tumor and stromal cells, stimulates the proliferation of endothelial cells, leading to new vessel formation, often accompanied by impaired tissue perfusion and increased vascular permeability 59. VEGF also decreases the expression of important molecules, such as VCAM-1, on the cell surface of the endothelium, ultimately preventing T cells from migrating to the TME 59. Another mechanism through which tumor endothelial cells can inhibit T-cell migration is modulation of immune cell activity or viability. IL-10, prostaglandin E2 (PGE2), and VEGF induced FasL upregulation in tumor endothelial cells to kill tumor-associated T cells, and anti-FasL attenuated this killing effect. Acetylsalicylic acid (ASA), which inhibits COX and PGE2 activity, and anti-VEGF antibodies promoted CD8+ T lymphocyte infiltration in the TME and improved prognoses 60. Furthermore, pericyte abnormalities and inadequate coverage prevent the maintenance of endothelial cell integrity, resulting in the dysfunctional leakage and flow characteristic of tumor vasculature 56, 61. However, other structures can promote the translocation of CD8+ T lymphocytes from blood vessels to tumor sites. The formation of high endothelial venules (HEVs) and related tertiary lymphoid structures (TLSs) facilitates T-cell migration to the TME and is often associated with better prognoses 46, 62.

【21】In addition, impaired vascular tight junctions and increased permeability result in the promotion of hypoxia, acidosis and necrosis, which inhibit immune effector T-cell functions and antitumor immunity 56. As a hallmark of cancer, hypoxia is caused by increased oxygen demand due to tumor cell proliferation and inadequate blood supply due to angiogenesis 63. Hypoxia-inducible factor 1 (HIF1) is a key transcription factor activated by hypoxia 64. Hypoxia inhibits T-cell infiltration in several ways. First, hypoxia promotes the recruitment of immunosuppressive cells to the TME 65. Second, hypoxia-induced CCL28 and VEGF promote angiogenesis and affect T-cell trafficking 56, 66. Finally, the expression of two ectonucleotidases, CD39 and CD73, can be upregulated in tumors in response to hypoxia and transforming growth factor-β (TGFβ) 67. CD39 and CD73 catalyze the sequential conversion of ATP to extracellular adenosine (ADO) 68. ADO binds to the adenosine A2A receptor (A2AR) and inhibits the production of cytokines such as IL-2 and the development and proliferation of T cells 69. The inhibition of A2AR increased T-lymphocyte infiltration and led to improved tumor control in mouse melanoma models, suggesting a potential effect of the ADO signaling pathway in promoting T-cell exclusion 70. In addition, ADO can weaken antitumor immunity by inhibiting the effector functions of NK cells and DCs and by promoting the recruitment and polarization of MDSCs and Tregs 71.

【22】The immunosuppressive microenvironment at tumor sites, including dense stroma and immunosuppressive cells and factors, can prevent T-cell priming and infiltration in “cold tumors”. TGFβ is a potent immunosuppressive cytokine that promotes immune escape and blocks the acquisition of the TH1-effector phenotype 72. CAFs, which are abundant in the TME, are the main producers of TGFβ. Increased TGFβ production by CAFs is associated with T-cell exclusion from the tumor and a poor response to atezolizumab 73. TGFβ limits the proliferation of CD4+ T lymphocytes by inhibiting the production of IL-2 and induces the conversion of naïve CD4+ T lymphocytes into Tregs 74, 75. TGFβ also negatively affects DC differentiation and antigen-presenting functions, which interfere with T-cell priming 76. In summary, TGFβ hinders antitumor immunity by affecting T-cell differentiation and function and preventing T-cell infiltration into tumors.

【23】Tryptophan metabolism is often dysregulated in a broad range of cancers and is associated with immune resistance. Indoleamine 2,3-dioxygenase (IDO) in tumor cells converts the essential amino acid tryptophan into kynurenine, which blocks the priming of T lymphocytes and facilitates the development of Tregs 77. IDO also recruits and activates MDSCs and inhibits the accumulation of tumor-specific T lymphocytes in tumors 78. IDO inhibitors such as epacadostat and navoximod have been used in combination with ICIs with promising results in clinical trials 79. However, the failure of epacadostat in combination with pembrolizumab in the phase III clinical ECHO-301 study indicates that the effectiveness of drugs targeting IDO needs to be further considered 80.

【24】CAFs are key cellular components in the tumor stroma and can promote tumor growth 81. CAFs are predominantly located at the infiltrating edges of tumors, regulating tumor metastasis and influencing angiogenesis by synthesizing and remodeling the extracellular matrix (ECM) and producing cytokines, and transforming tumor margins into immune “cold” zones 52, 82. CAFs led to immunosuppression and T-cell exclusion through several mechanisms. First, CAFs produce extracellular matrix that forms a physical barrier to prevent T-cell infiltration into the tumor area 83. Second, CXCL12 produced by CAFs has been shown to inhibit T-lymphocyte infiltration within tumors in a pancreatic cancer model 84. Third, CAFs can also reduce T-cell responses and exert immunosuppressive effects through the production of TGFβ and IL-6 82. Reprogramming CAFs is an effective strategy to “normalize” the TME. This strategy reduces ECM levels, decompresses blood vessels, and increases the degree of T-cell penetration to improve cancer treatment 85.

【25】In addition, TAMs exclude T cells from the tumor by regulating the ECM and mediating the nitration of CCL2 and CCL5 86, 87. TAMs affect T-cell recruitment by promoting abnormal angiogenesis through the production of VEGF and matrix metalloproteinase-9 (MMP9) 88. Cytokine colony-stimulating factor-1 (CSF-1) and CSF-1R interactions are capable of promoting myeloid cell differentiation towards an immunosuppressive M2 macrophage phenotype. Targeting TAMs with CSF1R inhibitors reduces the number of TAMs and increases the infiltration of effector lymphocytes such as CD8+ T cells 89.

【26】Tumor cells are typically characterized by a high rate of glucose uptake and active glycolysis, even in the presence of oxygen. This phenomenon is known as the “Warburg effect”. In this process, glucose is rapidly consumed and the abundance of lactate in the TME increases. The glucose-deficient, lactate-rich TME exerts metabolic stress on infiltrating T cells, leading to local immunosuppression and ICI resistance 77. Glucose deprivation in the TME metabolically mediates T cell hyporeactivity, inhibits mTOR activation, and reduces glycolytic capacity and IFN-γ production 90. In addition, glycolytic activity and T-cell infiltration are negatively correlated in a variety of tumors 91-93. Consistent with this observation, high glucose-transporter 1 (GLUT-1) expression in renal cell carcinoma are associated with low infiltration of CD8+ T cells 92. These results suggest an association of glycolytic (Warburg) tumors with a noninflamed T-cell phenotype. Interestingly, in addition to tumor cells, stromal cells, such as CAF and TAM, can also promote lactate accumulation in the TME through the so-called “Reverse Warburg effect” 91. Targeting glucose metabolism and lactate production in tumor and stromal cells, such as inhibition of LDH-A, may be an effective strategy to promote T-cell infiltration 94. Lactate accumulation and acidification of the TME suppress antitumor immunity. Lactate-induced acidosis impairs the differentiation of monocytes to DCs and inhibits the antigen-presenting function of DCs, which in turn inhibits T-cell activation 95. High concentrations of lactate and acidification in the TME inhibit monocarboxylate transporter 1 (MCT1)-mediated lactate release from T cells and suppress the proliferation of T cells that utilize aerobic glycolysis 96. In addition, lactate inhibits the chemotaxis and antitumor activity of CTLs and promotes tumor immune escape 96. Inhibition of lactate production or restoration of physiological pH of the TME can reverse the inhibitory effect of lactate on antitumor immunity. For example, neutralizing tumor acidity with sodium bicarbonate in combination with ICIs or adoptive cellular therapy (ACT) can effectively promote T-cell infiltration and improve antitumor responses in a variety of mouse tumor models 97.

【27】In addition to glucose, metabolic competition between tumors and immune cells includes amino acids and fatty acids. For example, the high rate of cholesterol esterification in tumors inhibits T cell receptor (TCR) aggregation and immune synapse formation 77. The cholesterol esterification key enzyme ACAT1 inhibitor avasimibe can promote the proliferation of CD8+ T cells and exhibit good antitumor effects 98. New studies have also confirmed that inhibition of PCSK9, a key protein regulating cholesterol metabolism, upregulates MHC-I levels on the surface of tumor cells, increases intratumoral infiltration of CTLs, and synergistically inhibits tumor growth with anti-PD1 antibodies 99. Considering the interaction between tumor metabolism and immune cell metabolism, navigating metabolic pathways to reduce metabolic stress on T cells is a promising strategy to improve the efficacy of immunotherapy.

【28】ICIs have revolutionized cancer treatment by activating T-cell-based antitumor immunity. However, a significant number of patients show a poor response to ICIs due to the multiple mechanisms mediating T-cell exclusion. Several approaches have been shown to drive T cells into tumors. These approaches “fire up” “cold tumors” to improve the efficacy of ICIs (Figure 4 and Table 3).

Approaches to turn a “cold tumor” into a “hot tumor”. Some representative approaches that lead to increased T-cell infiltration and improved efficacy of immune checkpoint inhibitors are highlighted here. (A) Oncolytic viruses, local thermal ablation therapy (e.g., radiofrequency ablation), chemotherapy, and radiotherapy are all capable of inducing immunogenic cell death (ICD) to promote T-cell priming and activation. Local administration of immune adjuvants such as TLR agonists promotes the activation of dendritic cells (DCs). Epigenetic modification inhibitors can promote T-cell priming by increasing the expression of tumor antigens and by restoring antigen processing and presentation mechanisms. (B) Cancer vaccines and adoptive cellular therapies, such as CAR-T cells, can promote the expansion of tumor-specific T lymphocytes. (C) Intrinsic oncogenic pathway inhibitors, epigenetic modification inhibitors, antiangiogenic therapies, TGFβ inhibitors, and CXCR4 inhibitors promote T-cell trafficking and enable T cells to infiltrate the tumor more effectively.

【29】Innate immune sensing pathways play critical roles in the development of antitumor immunity. The PRR family includes Toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-I-like receptors (RLRs), and C-type lectin receptors (CLRs) 27. When TLRs are stimulated, DCs can produce a variety of proinflammatory cytokines, including tumor necrosis factor (TNF), IL-1, and type I IFNs 27. DCs are the main sources of type I IFNs, which facilitate the expression of MHC-I on the surface of tumor cells and the maturation of DCs, thereby promoting T-cell priming 100.

【30】In contrast to therapies such as vaccines and CAR-T cells, immune adjuvants harness the endogenous antigen repertoire in the tumor and have been used to enhance the immune response for the treatment of malignant tumors. Local administration of the TLR7/8 agonist imidazoquinoline with coupled nanoparticles significantly activated the DCs of secondary lymphoid organs, upregulated the expression of MHC-II, CD40, and CD86 on their surface, and expanded the number of tumor-specific CD8+ T lymphocytes, which inhibited tumor growth 101. In clinical trials of patients with advanced malignant melanoma, combined treatment with the TLR9 agonists SD-101 and pembrolizumab resulted in increased type I IFN production and CD8+ T-cell infiltration and potentially improved clinical efficacy 102.

【31】As DNA receptors in the cytoplasm, cyclic GMP-AMP synthase (cGAS) in DCs, macrophages, and other immune cells recognizes aberrant DNA in the cytoplasm and catalyzes the formation of cGAMP, which subsequently activates the STING signaling pathway 103. Activation of the STING signaling pathway mediates the expression of proinflammatory cytokines (e.g., type I IFNs) and chemokines (e.g., CXCL10) in a TBK1-IRF3-dependent manner, thereby initiating the antitumor immune response 104. The systemic cGAMP mimetic SR-717 activated the STING signaling pathway, promoted CD8+ T-cell, NK cell, and CD8α+ DC activation, and significantly inhibited tumor growth 105. SR-717 also induced PD-L1 expression in a STING-dependent manner, revealing the significance of the combination of STING agonists and ICIs for tumor treatment. In mouse tumor models with a low response to PD-1 blockade, the combination of PD-1 blockade and the STING agonist MSA-2 increased the infiltration of tumor CD8+ T lymphocytes and better inhibited tumor growth 106.

【32】OVs are now being recognized as emerging therapeutics with potent anticancer activity. In addition to selective tumor lysis, they can activate both innate and adaptive immune responses, resulting in alterations in the TME. First, lysis of tumor cells by OVs induces immunogenic cell death (ICD), leading to a massive release of intracellular TAAs, PAMPs, and DAMPs 107. Three DAMPs are released during ICD: passively released high mobility family protein B1 (HMGB1), actively secreted extracellular ATP, and cell surface-expressed CRT 108. These DAMPs act as adjuvants to promote DC uptake and cross-present tumor antigens to T lymphocytes in DLNs. OVs also improve the function of DCs by stimulating their production of type I IFNs. Immune adjuvants interact with tumor antigens in tumor residues and act as individualized in situ vaccines to promote T-cell priming 109. Second, OVs stimulate the production of CXCL9 and CXCL10 and upregulate the expression of selectins and integrins, providing key signals for T-cell trafficking. In addition, the degradation of the ECM by OVs disrupts the physical barrier to T-cell infiltration 109. OVs can also deplete the immunosuppressive effects of CAFs, TAMs and MDSCs, significantly altering the TME 109.

【33】Talimogene laherparepvec (T-VEC) was first shown to be effective as an oncolytic virotherapy for melanoma 110. Combination therapy with T-VEC and pembrolizumab increased CD8+ T-lymphocyte infiltration, IFN-γ expression and the therapeutic effect of PD-1 blockade in patients with advanced malignant melanoma 111. Similar effects have been observed with combination therapy of coxsackievirus and pembrolizumab 112. These studies suggest that combination therapy with OVs and ICIs can improve CD8+ T-cell infiltration and activation and help to overcome the resistance of cancer to ICIs in patients. Combined with T-cell therapy, promoting T-cell proliferation and infiltration into the local TME is a potential direction for the development of OVs.

【34】It was previously thought that chemotherapy and radiotherapy exert their antitumor effects by directly killing tumor cells. However, accumulated evidence suggests that tumor suppression by chemotherapy and radiotherapy also relies on stimulating the immune system. When radiotherapy is administered to a local tumor, distant tumors outside the irradiated field also shrink. This phenomenon is termed the “abscopal effect”, which indicates the significance of the immune system in radiotherapy-mediated antitumor responses 113. After radiation therapy causes damage to tumor cells, ROS and endoplasmic reticulum (ER) stress mediate cellular stress and lead to ICDs 114. This cascade promotes DC activation, increases the production of TNFα and IL-1 and produces endogenous cancer vaccines in vivo 114. After radiation therapy, endothelial cells express ICAM1, VCAM1, and E-selectin, which facilitate the attraction of immune cells 114. Radiation also promotes the trafficking of effector T lymphocytes to the tumor site by inducing tumor cells to express and release chemokines (e.g., CXCL10 and CXCL16) 115. However, considering the adverse effects of radiation therapy, it is necessary to optimize the radiation dose and fractionation levels during radiation therapy. Fractionated radiotherapy at individual doses of less than 8-10 Gy helps to induce sufficient ICD without increasing hypoxia or immunosuppression, inducing a de novo antitumor response 115. In preclinical studies, stereotactic body radiotherapy (SBRT) increased effector T-cell infiltration in tumors and DLNs and was associated with higher survival rates 116.

【35】In addition to radiotherapy, many chemotherapeutic agents can exert their immunostimulatory effects by enhancing immunogenicity and increasing T-cell infiltration 117. ICD inducing chemotherapy has been shown in various mouse models to contribute to the transformation of “cold tumors” to “hot tumors” in response to ICIs 118-120. New immunotherapies that reduce the toxic side effects of systemic chemotherapy and enhance the immunogenicity induced by chemotherapeutic agents deserve further exploration. A new cocktail therapy involves local chemoimmunotherapy by mixing chemotherapeutic agents with immune adjuvants and alginate (ALG). The ICD-inducing chemotherapeutic agents produced tumor vaccines in situ in response to adjuvant stimulation. In situ gelation of the drug adjuvant ALG enables slow release of the drug, thereby reducing systemic toxicity. The combination of ICIs further amplifies the immune response and inhibits tumor metastasis and recurrence 121.

【36】Image-guided thermal ablation has been developed as a promising method for the treatment of solid tumors. Currently, the commonly used thermal ablation methods include radiofrequency ablation (RFA), laser ablation (LA), microwave ablation (MWA), and high-intensity focused ultrasound (HIFU) ablation. RFA is widely used in the treatment of solid tumors, especially hepatocellular carcinoma (HCC). RFA utilizes the conversion of radiofrequency alternating current into heat to ablate the tissue surrounding the needle electrode and stimulate tumor-specific T-cell responses 122. However, problems with incomplete ablation and tumor recurrence are drawbacks of RFA. The combination of the tyrosine kinase inhibitor sunitinib and RFA improves HCC treatment. RFA caused the release of TSA in situ in tumors but caused the upregulation of PD-1 expression in T lymphocytes, which was related to the exhaustion of CD8+ T lymphocytes. Sunitinib inhibition of hepatocyte growth factor (HGF) inhibited the upregulation of PD-1 expression in tumor T lymphocytes. Inhibition of VEGF by sunitinib also promoted DC activation and inhibited tumor angiogenesis. Combination therapy ultimately led to a remarkable increase in CD8+ T lymphocytes and DCs as well as a decrease in Tregs, thus overcoming the drawbacks of monotherapy 123. In addition, HIFU, as a new minimally invasive ablation therapy, has become a hotspot of cancer treatment. HIFU delivers acoustic energy to the target tissues in a noninvasive and precise manner, generating high temperatures that cause coagulative necrosis of the tumor tissue. HIFU promotes ICD and the activation of T cells 124. HIFU also promotes antigen transfer to lymph nodes and T-cell migration to tumors through mechanical destruction of the mesenchyme 124.

【37】ACT enhances the immune response of effector T cells to cancer, including tumor-infiltrating lymphocytes (TILs) and CAR-T cells 6. In TIL therapy, tumor-infiltrating lymphocytes are isolated from cancer patients, expanded in vitro, and then reinfused into the patient. However, due to the small number of infiltrating lymphocytes or the downregulation of MHC molecules, TILs are only used for the treatment of a few tumor types, such as malignant melanoma 125. CAR-T cells involve genetic modification of T lymphocytes to express the CAR to target tumor cells expressing a specific antigen 6. For example, CD19-specific CAR-T cells have become the gold standard for the treatment of B-cell malignancies. In addition, CAR-T cell therapy for leukemia and lymphoma was approved by the FDA in 2017 6. In contrast to TILs, CAR-T cells are not limited by the MHC and can further enhance the immune response to tumors through the addition of costimulatory domains (e.g., CD28, OX40, and 4-1BB) 6. This strategy leverages the direct recognition of tumor antigens by CAR-T cells and has the potential to treat “cold tumors” lacking pre-existing T-cell infiltration. CAR-T cells expressing IL-7 and CCL19 increased DC and T-cell infiltration in mouse solid tumor tissues and showed potent antitumor effects 126. In addition, the use of FLT3L-secreting CAR-T cells and immune adjuvants led to similar results and induced host T-cell antigen epitope spread 34. However, CAR-T cells exhibit limited clinical efficacy in solid tumors due to tumor antigen heterogeneity and insufficient infiltration into tumors 6. Additionally, the immunosuppressive microenvironment limits the tumor-killing effect of CAR-T cells, which makes it necessary to combine CAR-T cells with ICI therapy.

【38】Therapeutic vaccines such as peptide and tumor cell vaccines, nucleotide vaccines encoding new epitopes, and dendritic cell vaccines have been encouraging clinical advances. Therapeutic vaccines expand the pool of tumor-specific T cells, increase the transport of T lymphocytes to tumor areas, and have become emerging modalities for immunotherapy 127. Sipuleucel-T, the first therapeutic cancer vaccine licensed by the FDA, has been used in the treatment of castration-resistant prostate cancer. Sipuleucel-T consists of the fusion protein PA2024 constructed from both prostatic acid phosphatase (PAP) and GM-CSF and autologous DCs, which enhance the antitumor effect 128. Tumor neoantigens are highly tumor-specific and immunogenic. The combination of the personalized neoantigen vaccine NEO-PV-01 with nivolumab significantly prolonged progression-free survival in an Ib clinical trial for patients with advanced malignant melanoma, NSCLC and bladder cancer. A neoantigen-specific T-cell response, T-cell trafficking to tumors and induction of epitope spread were also observed 129. The combination of the mRNA personalized cancer vaccine RO7198457 and atezolizumab showed clinical benefit in a phase Ib clinical trial for patients with advanced solid malignancies (NCT03289962). The vaccine induced a neoantigen-specific T-cell response in 77% of patients. This outcome demonstrates that the use of a personalized cancer vaccine combined with an immune checkpoint blockade can generate a specific immune response in patients. However, the complex operation, cumbersome process and high price remain limiting factors for the widespread use of personalized cancer vaccines in cancer treatment.

【39】The use of oncogenic pathway inhibitors helps reverse the inherent T-cell exclusion from tumors. PAK4 is abundantly expressed in “cold tumors” and plays a key role in the WNT/β-catenin pathway. Knocking down PAK4 or applying the PAK4 inhibitor KPT-9274 in a mouse tumor model enhanced CTL infiltration in tumors and improved the therapeutic efficacy of a PD-1 blockade 130. However, the efficacy of treatments targeting WNT remains controversial. For example, endogenous inhibitors of the WNT pathway, such as some proteins of the dickkopf (DKK) family, have a role in promoting tumor immune escape and are associated with a poorer prognosis in some cancers 131, 132. DKK2 inhibits WNT-β-catenin signaling by binding to the cell surface receptors LRP5 and LRP6 of the WNT pathway 133. DKK2 expression is upregulated in human colorectal cancers (CRCs) and promotes tumor progression by inhibiting the activation of NK cells and CD8+ T cells 131. This challenges the notion that inhibition of the WNT pathway will improve immunotherapy. Furthermore, recent studies have shown that activation of the WNT pathway in endothelial cells promotes T-cell infiltration into tumors and enhances the effectiveness of immunotherapies such as the ACT, suggesting that there is still a need to further investigate the feasibility of using WNT inhibitors as immune adjuvants 134. Indeed, clinical data using inhibitors of the WNT/βcatenin pathway do not support completely its putative function to boost immunotherapy in the clinic.

【40】Activation of the PI3K-AKT pathway is associated with the inhibition of CTL infiltration and function. PI3Kβ inhibitors inhibit the activation of the AKT pathway in PTEN-deficient melanoma cell lines and enhance T-cell-mediated killing 38. The combination of PI3Kβ inhibitors and ICIs significantly increased the number of infiltrating T lymphocytes in murine tumor models 38.

【41】RAS was considered as an “undruggable” target in the past 135. Monotherapies targeting the RAS oncogene have faced limited efficacy due to multiple mechanisms, such as feedback reactivation of the RAS downstream pathway 39. However, recent studies have found that ARS-1620, a small-molecule inhibitor that specifically targets KRAS-G12C mutants, significantly inhibited the growth of KRAS-G12C tumors 136. This evidence suggests a new therapeutic avenue to inhibiting mutant RAS. In addition, MEK inhibitors in combination with ICIs led to an increase in tumor-infiltrating T cells and a decrease in the percentage of MDSCs, which in turn significantly inhibited tumor growth in TP53/KRAS-driven lung cancer mouse models 137. Oncogenic mutations of BRAF activate the RAF-MEK-ERK (MAPK) pathway. Inhibition of BRAF or MEK inhibits the production of inhibitory cytokines (e.g., IL-6, IL-10, and VEGF) or enhances the expression of melanocyte differentiation antigen, thereby promoting melanoma recognition by T cells 138, 139. Three variant kinase inhibitors targeting MEK, cobimetinib, trametinib, and binimetinib, are clinically approved for therapeutic use in BRAF V600 mutant melanoma 135.

【42】CDk4/6 can bind to cyclin D, which enables cells to enter S-phase through the RB-EF2 pathway and promotes tumor cell proliferation. Treatment of CT26 syngeneic mouse tumors with the CDK4/6 inhibitor abemaciclib led to an increase in tumor-infiltrating T lymphocytes, and a significant upregulation of T-cell activity, as evidenced by the increased expression of T-lymphocyte activation markers (e.g., IFNG, GZMB, CCL4 and CCL5). Abemaciclib also led to enhanced antigen presentation and had a synergistic effect when applied with anti-PD-1 therapy 44.

【43】Epigenetic therapies can transform tumors from the immune “cold” state to the immune “hot” state through a variety of mechanisms. Epigenetic drugs can enhance the expression of multiple chemokines, such as CXCL9, CXCXL10, and CCL5, and promote T-cell trafficking to tumors 48, 49, 51. Epigenetic therapy can also induce ERVs and suppress MYC signaling, thereby enhancing the expression of type I IFNs and related chemokines 51. In addition, epigenetic therapies can enhance tumor immunogenicity by increasing the expression of tumor antigens such as CTA and by restoring MHC-I antigen processing and presentation mechanisms 140, 141. The DNMT inhibitor guadecitabine upregulated MHC-I expression in breast tumor cells, enhanced IFN-γ responses, and promoted T-cell recruitment to tumors. In addition, guadecitabine had a synergistic effect with an anti-PD-L1 antibody 142. This outcome suggests the feasibility of combining epigenetic inhibitors with ICI strategies for future clinical application. A variety of epigenetic drugs have been approved by the FDA, such as azacitidine and decitabine (DNMT inhibitors), tazemetostat (an EZH2 inhibitor), and entinostat and vorinostat (HDAC inhibitors) 143.

【44】Persistent angiogenesis caused by a dysregulated balance between pro-and antiangiogenic signals is one of the hallmarks of tumors 144. Antiangiogenic therapy (AT) structurally and functionally overcomes tumor vascular abnormalities, improves tissue perfusion, and increases the infiltration of immune effector cells. AT-mediated immune reprogramming in turn improves vascular normalization, thereby creating an enhanced positive feedback loop 56, 145. Bevacizumab is the first FDA-approved angiogenesis inhibitor. Increased infiltration of tumor-specific T lymphocytes was observed after combination therapy with bevacizumab and atezolizumab in patients with metastatic renal cancer. In addition, combination therapy resulted in downregulation of the expression of vascular signature genes (e.g., ANGPT2 and CD31) and upregulation of CD8+ T effector genes (e.g., CD8A, GZMB, and IFNG) and MHC-I, as well as chemokines (e.g., fractalkine) 146. These results imply that the increase in tumor-specific T-cell infiltration may be due to enhanced lymphocyte trafficking mediated by the combination therapy. In addition, in the phase III IMbrave150 trial for patients with unresectable hepatocellular carcinoma, treatment with atezolizumab in combination with Bevacizumab significantly improved overall survival and progression-free survival outcomes compared with the results of sorafenib treatment 147. Given the relationship between vascular normalization and immune reprogramming, combination therapy is expected to further reverse the immunosuppressive microenvironment and improve the efficacy of immunotherapy.

【45】Considering the immunosuppressive function of TGFβ, therapy based on the inhibition of TGFβ has been validated as an effective approach to promote T-lymphocyte infiltration. TGFβ is related to a lack of immune response in the noninflamed T-cell phenotype. In a mammary cancer mouse model with the immune-excluded phenotype, combined treatment with anti-PD-L1 and anti-TGFβ antibodies significantly reduced tumor burden and increased tumor-infiltrating T cells, especially CD8+ T effector cells 73. Galunisertib, a small molecule that inhibits the activity of TGFBR1 kinase, has been the most widely tested compound. Galunisertib treatment increased T-cell infiltration and improved susceptibility to checkpoint therapy in a mouse colorectal model 72. TGFβ impedes the generation of in situ tumor vaccines after radiotherapy. Treatment with the 1D11 antibody, which blocks systemic TGFβ activity, enhanced the initiation of T-cell responses to endogenous tumor antigens after subcutaneous tumor irradiation 148.

【46】CXCR4 is a receptor for CXCL12, which is overexpressed in a wide range of tumors. The CXCL12-CXCR4 axis plays an indirect role in the sequestration of CTLs from the tumor area to reduce CTL infiltration and mediates the infiltration of immunosuppressive cells into tumors 52. In the PDAC model, inhibition of CAF-mediated CXCL12/CXCR4 axis with the CXCR4 inhibitor AMD3100 promoted T-cell accumulation and cancer regression 84. Previous studies have shown that immunotherapies such as pembrolizumab were not effective against “cold tumors” such as pancreatic cancer. However, in the COMBAT trial, the synergistic treatment of metastatic PDAC with the CXCR4 antagonist BL8040 and pembrolizumab increased tumor-infiltrating CD8+ effector T lymphocytes, reduced the density of MDSCs in tumors, and reduced the number of circulating Tregs 149. These results reveal that regulating certain chemokines facilitates the homing of tumor-specific T lymphocytes to the tumor and reverses immune resistance.

【47】A variety of nanomedicines combined with immunotherapy contribute to the transition from “cold tumors” to “hot tumors” (Figure 5). Nanomedicines have three different targeting pathways: tumor cells, the TME, and the peripheral immune system 150. Nanomedicine-based targeted tumor therapy includes passive and active targeting. Passive targeting can promote selective accumulation of nanomedicines in tumors through EPR effects 151. However, recent studies have suggested that the passive targeting ability of nanomaterials may be associated with transcytosis 152. Active targeting involves the use of targeted ligands (e.g., peptides, antibodies and transferrin) that specifically recognize specific receptors expressed by tumor cells 15.

Improving T-cell infiltration with nanomedicines. Nanomedicines have three different targeting pathways: tumor cells, the TME, and the peripheral immune system. (A) Multiple approaches including photothermal therapy (PTT), photodynamic therapy (PDT), magnetic hyperthermia (MH), and high-intensity focused ultrasound (HIFU) can induce ICD by promoting the release of tumor antigens and damage-associated molecular patterns (DAMPs). The released DAMPs act as adjuvants to enhance the immunogenicity of the tumor and, together with the released tumor antigens, promote dendritic cell (DC) activation and T-cell priming. (B) When targeting the TME, nanomedicines inhibit immunosuppressive cells and immunosuppressive molecules (e.g., TGFβ) and enhance the activity of T cells. (C) When the peripheral immune system is targeted, nanomedicines are engineered to augment tumor antigen presentation and T-cell priming in lymph nodes. Adapted with permission from 150, copyright 2019 American Chemical Society.

【48】Nanomedicines targeting tumor cells can induce ICD and enhance the tumor-immunity cycle 150, 153. For example, doxil, a PEGylated liposome of the chemotherapeutic drug doxorubicin, promoted DC and CD8+ T cell proliferation via ICD and inhibited Treg infiltration 154. Doxil was also synergistic with ICIs (anti-PD-1 and anti-CTLA-4) and showed higher efficacy than free doxorubicin 154. When targeting the TME, nanomedicines inhibit immunosuppressive cells (e.g., M2 TAMs and MDSCs) and immunosuppressive molecules (e.g., TGFβ and ADO), and they can also augment the activity and function of effector immune cells (e.g., macrophages and CTLs) 150, 155. The TGFβ receptor inhibitor SB525334 was loaded onto liposomes targeting ACT T cells. This nanomedicine inhibited TGFβ expression and promoted T-cell activation as well as tumor regression in melanoma mice 156. Loading liposomes with IL-2 and agonistic anti-4-1BB enhanced the tumor infiltration of CTLs as well as cytokine production and granzyme expression 157. When targeting the peripheral immune system, nanomedicines are designed to promote antigen presentation and CTL production in DLNs. Nanomedicine-based vaccines improved antigen delivery to lymph nodes, promoted antigen cross-presentation, and increased CTL activation levels 150, 158, 159. Furthermore, nanomedicines have been engineered to directly promote T-cell priming by replacing APCs in secondary lymph nodes. The use of biomimetic magnetosomes as artificial APCs was characterized by magnetic nanoclusters encapsulated by leukocyte membranes and modified to stimulate signals on the membranes. Artificial APCs not only expanded and stimulated CTLs, but also guided reinfused CTLs efficiently into tumor tissues, thus inhibiting tumor growth 160.

【49】PT, including photothermal therapy (PTT) and photodynamic therapy (PDT), has been developed as a potential treatment for solid tumors, especially superficial tumors. PTT uses photothermal agents (e.g., organic nanoparticles, gold nanoparticles and graphene oxide) with photothermal conversion capabilities to absorb near-infrared (NIR) lasers and convert them into heat energy to kill tumor cells 15. PDT involves the use of a laser to irradiate the tumor into which photosensitizers had been delivered to activate the photosensitizers and produce cytotoxic ROS. It can cause DNA damage in the nucleus and thus induce tumor cell death 15. Compared to other ablation modalities (e.g., radiofrequency ablation and microwave ablation), PT is more selective and less toxic to surrounding tissues due to the accumulation of photosensitizers in the tumor and the controllability of the light.

【50】PTT and PDT induce ICD through thermal and chemical damage, respectively, and enhance the infiltration of CTLs and the immunotherapeutic response 15. PDT based on a layer-by-layer Apt/ PDGsˆs@pMOF nanoplatform enhanced the immunogenicity of triple-negative breast cancer in mice, selectively suppressed MDSCs, and promoted the transition to “hot tumors” 161. However, a single PT treatment shows limited efficacy because of limited light penetration, a heat-shock response due to the PTT, and the dependence of PDT on oxygen. The combination of PDT and PTT can achieve promising synergistic antitumor effects. A hybrid nanoporphyrin (Pp18-Lips)-mediated synergistic PTT/PDT caused an increase in tumor-infiltrating CTLs and inflammatory cytokines (e.g., TNF-α and IFN-γ) and a decrease in Tregs. Synergistic PDT/PTT produced a stronger antitumor immune response and stronger tumor suppression than PDT or PTT alone 162. PT is also synergistic with immunotherapy, as PT enhances the immunogenicity of the tumor by mediating ICD, while immunotherapy enhances the “abscopal effect” of the treatment. PTT-mediated GOP@aPD-1 nanoparticles efficiently delivered anti-PD-1 to melanoma cells and combined ICI treatment with tumor-targeted PTT. This combination resulted in increased CD8+ T-cell infiltration, elevated efficacy of the PD-1 blockade in mouse melanoma, and inhibition of tumor growth 163.

【51】MH refers to the selective heating of tumors by converting magnetic energy into thermal energy through the hysteresis and relaxation effects of magnetic nanoparticles (MNPs) in the presence of an alternating magnetic field (AMF) 164. In addition to killing tumor cells with heat, MH also induces ICD, showing great potential in cancer therapy. Compared to PT and thermal ablation therapy, MH has no penetration depth limit and enables more effective targeting and more precise control of the heating temperature. The iron oxide nanomedicine ferumoxytol promoted the polarization of macrophages into the proinflammatory M1 phenotype. This switch induced the apoptosis of tumor cells, as indicated by increased caspase-3 cleavage 165. Another typical example is ferrimagnetic vortex-domain iron oxide nanorings (FVIOs), which have excellent nanomagnetic properties. FVIO-mediated mild MH induced the expression of CRT on the surface of 4T1 breast tumor cells and promoted T-cell activation. FVIOs caused a significant increase in CTL infiltration along with a decrease in MDSCs and were synergistic with anti-PD-L1 antibodies 166.

【52】The combination of HIFU and immunotherapy achieved significant therapeutic efficacy. Compared to immunotherapy alone or HIFU alone, the combination of HIFU and the TLR agonist CpG in mouse melanoma enhanced antigen cross-presentation in DLNs, release of type I IFNs, and expression of genes relevant to T-cell priming and stimulation (e.g., Eomes, Prf1 and Icos) 167. However, HIFU remains under-researched and there is a lack of studies demonstrating tumor control with HIFU monotherapy 124. The current state of the field suggests that HIFU can promote T-cell priming and tumor regression, but induction of additional immune adjuvants may be necessary. The killing of tumor cells by HIFU has also been linked to cavitation effects, which can efficiently aggregate energy. A recent study showed that direct injection of microbubbles and plasmid DNA encoding IFN-β into tumors in a mouse model followed by application of low-frequency ultrasound (250 kHz) to break up the tumors was shown to remove a substantial number of tumor cells and simultaneously achieve massive infiltration of CTLs. The remaining tumor cells also formed membrane pores, allowing gene transfection of the cells and triggering antitumor immune responses 168.

【53】Considering the relevance of T-lymphocyte infiltration in tumor sites to the prognosis of ICI therapy, the cause of the absence of a T-cell response is crucial to determine. In this review, we summarize various mechanisms that inhibit T-cell infiltration, such as defects in tumor antigen processing and presentation processes, endogenous oncogenic pathway activation, aberrant vasculature and chemokines, and TME suppression. Other known and unknown factors affecting T-cell infiltration, such as TLSs and the microbiome, remain to be evaluated. TLSs have a lymph node-like function and are relevant to T-cell infiltration into tumors and a good response to immunotherapy 169, 170. Exploring therapeutic strategies to enhance TLS formation and function may promote the activation of naïve T lymphocytes by DCs in close proximity to the tumor and improve the response to cancer immunotherapy. Compared to “cold tumors”, “hot tumors” are more responsive to ICI monotherapy. Thus, promoting the conversion of “cold tumors” to “hot tumors” through interventions can help reduce resistance to ICIs. In addition, we then discuss a variety of therapeutic measures to improve T-cell infiltration, such as oncogenic pathway inhibitors, anti-vascular therapy, ACT, vaccines, oncolytic viruses, and cytotoxic therapies. The combination of ICIs with these therapies reverses T-cell exhaustion, enhances the “abscopal effects” of therapy, and demonstrates incremental clinical efficacy. However, the optimal dose and sequence of administration of combination therapy needs to be further evaluated to optimize T-cell function, promote T-cell memory, and avoid overactivation 171. In addition, some issues still need to be addressed, such as the nonspecific distribution of drugs, and the treatment-induced systemic adverse effects.

【54】

With the development of nanotechnology, the nanomedicine and biomaterial-assisted local therapies offers new opportunities for the future. The use of nanomedicines improves drug precision and bioavailability, reduces immunotherapy-induced side effects, and enables selective accumulation of drugs in tumors through EPR effects and active targeting. As mentioned above, PTT, PDT, MH, and HIFU are all capable of inducing a de novo antitumor response, which is achieved through the induction of ICD. These approaches do not require prior knowledge of tumor antigens and induce the generation of endogenous personalized in situ vaccines. However, killing solid tumors by noninflammatory apoptosis or ablation does not make tumor cells sufficiently immunogenic. In contrast to apoptosis, pyroptosis is a proinflammatory form of cell death that leads to the release of a massive quantity of inflammatory molecules (e.g., IL-1β and IL-18), mobilizing a robust antitumor T-cell response (Figure 6) 172-174. The use of nanotechnology to induce pyroptosis increases the immunogenicity of tumor cells and may effectively improve T-cell infiltration in tumors 172. For example, Zhao et al designed biomimetic nanoparticles (BNPs) loaded with indocyanine green (ICG) and decitabine for photoinduced pyroptosis. Due to promoter methylation of the GSDME gene, GSDME expression is much lower in most tumor cells than in normal cells. As an inhibitor of DNA methylation, decitabine promotes caspase-3 cleavage to GSDME by upregulating GSDME expression, thus leading to tumor cell pyroptosis. Pyroptosis mediated by BNP results in the release of a large number of inflammatory molecules from tumor cells and induces DC maturation and T-cell activation in DLNs, demonstrating a robust immune response against primary and distant tumors 175.

Schematic illustration of pyroptosis and size-transformable nanoparticles. (A) Multiple nanomedicines regulate the expression of caspase proteins that mediate the pyroptosis process. Activated caspases cut gasdermin (GSDM) into two fragments: the C-terminal domain and the N-terminal domain. Following cleavage, the gasdermin-N domains result in cell swelling with big bubbles. Gasdermin-induced pyroptosis results in the release of a massive quantity of proinflammatory molecules and activation of T cells. (B) Use of size-transformable nanoparticles to prolong the circulation time and realize deep penetration.

【55】However, certain challenges to nanomedicine use must be overcome, such as short blood circulation time, and insufficient penetration and accumulation in tumor tissues. The use of size-transformable nanoparticles in phototherapy or chemotherapy may achieve deep penetration of nanomedicines and improve T-cell infiltration into tumors 176, 177. Nanocarriers with relatively large particle sizes utilize the EPR effect to prolong the circulation time of the nanomedicine and improve its accumulation in tumor tissue. Upon reaching the tumor site, the nanomedicine undergoes size transformation in response to pH or enzymes and releases transformed small nanoparticles that exhibit effective tumor tissue penetration and can be thus efficiently internalized by tumor cells.

【56】In addition, the use of small molecular weight nanobodies in diagnostic imaging allows for a more convenient and complete assessment of the extent of T-cell infiltration in the TME, providing new ideas for achieving the integration of diagnosis and treatment of tumors. A better understanding of these aspects will be beneficial for guiding personalized cancer immunotherapy and extending the benefits of ICI therapy to a broader group of patients.

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