Intratumoral dose-specific GM-CSF modulates breast tumor oxygenation and anti-tumor immunity through macrophages

Background: Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) is a hematopoietic growth factor and adjuvant in cancer immunotherapy via stimulation of dendritic cells/APCs. However, GM-CSF has yielded inconsistent results and its role regarding in vivo modulation of macrophages remains underexplored. We previously demonstrated that 100ng “high-dose” intratumor (IT) GM-CSF ablated tumor blood vessels and worsened tumor hypoxia after 3 weeks through tumor-associated macrophage (TAM) soluble VEGFR-1 production in PyMT murine breast cancer. Here, we investigate a role for “low-dose” IT GM-CSF on tumor oxygen and the impact on immunotherapy response, TAMs/myeloid cells, and TILs relative to “high-dose”. Methods: We performed IT injections of dose-specific GM-CSF or saline controls and then evaluated phenotypic effects after 3 weeks. We used Electron Paramagnetic Resonance Oximetry to measure and image in vivo tumor oxygen in real-time, and fluorescent immunohistochemistry to assess tumor blood vessels. IT GM-CSF doses were tested in priming PyMT tumors for sensitization to ?PD1. We performed RNA Sequencing of TAM and CD8 TIL to observe transcriptional changes coupled with flow cytometry of peripheral blood monocytes, tumor myeloid, and TIL populations in immunology cold" PyMT tumors response to dose-optimized GM-CSF. Lastly, we assessed and compared effects of IT GM-CSF on TAM and TIL in an immunologically “hot” 4T1 breast cancer model. Results: 5ng IT GM-CSF significantly increased PyMT tumor oxygen without augmenting tumor growth and promoted tumor vessel health via increased pericyte coverage. Priming of PyMT tumors with 5ng IT GM-CSF (“low-dose”, hypoxia reduced) sensitized “cold” PyMT tumors to ?PD1, but this synergy was not observed with 100ng (“high-dose”, hypoxia exacerbated). Immunologically, 5ng GM-CSF did not increase monocyte mobilization or alter phenotypic marker expression on TAMs, but reduced hypoxic and inflammatory transcriptional programs in macrophages and CD8 TIL isolated from PyMT tumors. 100ng increased infiltration of myeloid cells and TAMs but these TAMs had reduced MHCII expression, suggesting support of immune-suppressive TAM under hypoxia. Some tumors exhibited increased CD8 polyfunctionality either dose suggesting mild CD8 TIL priming. On the other hand, 100ng in “hot” 4T1 tumors resulted in increased TAM MHCII and other immunostimulatory molecules with moderate increases in CD8 TIL polyfunctionality and exhausted PD1hiTIM3+ phenotype, indicating that GM-CSF may have opposing effects on macrophage modulation based on tumor immunological status. Overall design: 11 samples analyzed, with 2-3 replicates per cell type per treatment group (5ng GM-CSF) or saline control. Control samples are "saline treated". Macrophage "saline1" consists of 2 biological replicates pooled together due to low cell yield from 2 individual tumors.

背景：粒细胞-巨噬细胞集落刺激因子（Granulocyte-Macrophage Colony Stimulating Factor, GM-CSF）是一种造血生长因子，可通过刺激树突状细胞/抗原呈递细胞（dendritic cells/APCs）作为癌症免疫治疗佐剂。然而，GM-CSF的临床应用效果并不一致，其在体内调控巨噬细胞的作用仍未得到充分探索。我们此前的研究证实，在PyMT小鼠乳腺癌模型中，瘤内（intratumor, IT）注射100ng的“高剂量”GM-CSF可通过肿瘤相关巨噬细胞（tumor-associated macrophage, TAM）分泌可溶性血管内皮生长因子受体-1（soluble VEGFR-1），在3周后破坏肿瘤血管并加重肿瘤缺氧。本研究旨在探讨“低剂量”瘤内GM-CSF对肿瘤氧合的影响，以及相较于“高剂量”方案，其对免疫治疗响应、肿瘤相关巨噬细胞/髓系细胞及肿瘤浸润淋巴细胞（tumor-infiltrating lymphocytes, TILs）的作用。
方法：我们对小鼠进行剂量特异性GM-CSF或生理盐水对照的瘤内注射，并在3周后评估其表型效应。我们采用电子顺磁共振血氧测定法（Electron Paramagnetic Resonance Oximetry）实时测量并成像体内肿瘤氧合水平，同时通过荧光免疫组织化学（fluorescent immunohistochemistry）评估肿瘤血管状态。我们在经预处理的PyMT肿瘤模型中测试不同剂量的瘤内GM-CSF，以评估其对抗PD-1（anti-PD1）的增敏作用。我们对肿瘤相关巨噬细胞和CD8+肿瘤浸润淋巴细胞进行RNA测序（RNA Sequencing）以观察转录组变化，同时结合流式细胞术（flow cytometry）分析免疫“冷”型PyMT肿瘤中，经剂量优化的GM-CSF处理后外周血单核细胞、肿瘤髓系细胞及肿瘤浸润淋巴细胞群的变化。最后，我们在免疫“热”型4T1乳腺癌模型中评估并对比瘤内GM-CSF对肿瘤相关巨噬细胞和肿瘤浸润淋巴细胞的作用。
结果：瘤内注射5ng GM-CSF可显著提升PyMT肿瘤的氧合水平，且未促进肿瘤生长，并通过提升周细胞覆盖率改善肿瘤血管健康状态。以5ng瘤内GM-CSF（“低剂量”，可降低肿瘤缺氧）预处理PyMT肿瘤，可使“冷”型PyMT肿瘤对抗PD-1治疗产生增敏响应，但100ng“高剂量”方案（可加重肿瘤缺氧）未观察到该协同效应。免疫学层面，5ng GM-CSF未增加单核细胞募集，也未改变肿瘤相关巨噬细胞的表型标志物表达，但可降低从PyMT肿瘤中分离得到的巨噬细胞及CD8+肿瘤浸润淋巴细胞的缺氧相关及炎症相关转录程序表达。100ng GM-CSF可增加髓系细胞及肿瘤相关巨噬细胞的浸润，但此类肿瘤相关巨噬细胞的MHCII分子表达降低，提示在缺氧环境下可促进免疫抑制型肿瘤相关巨噬细胞的形成。两种剂量下部分肿瘤均出现CD8+ T细胞功能多样性提升，提示存在轻度的CD8+肿瘤浸润淋巴细胞致敏。另一方面，在免疫“热”型4T1肿瘤中使用100ng GM-CSF可提升肿瘤相关巨噬细胞的MHCII分子及其他免疫刺激分子的表达，同时轻度提升CD8+肿瘤浸润淋巴细胞的功能多样性，并诱导其耗竭型PD1hiTIM3+表型，这表明GM-CSF对巨噬细胞的调控作用可能因肿瘤免疫状态不同而产生相反效果。
整体实验设计：本研究共分析11份样本，每个细胞类型在5ng GM-CSF处理组或生理盐水对照组中设置2-3次生物学重复。对照组样本为“生理盐水处理组”。由于从2个独立肿瘤中获取的细胞产量较低，巨噬细胞组的“saline1”样本将2份生物学重复合并为一份进行分析。