Montefiore Medical Center, 1695 Eastchester Road, Bronx, NY, 10461, USA.
Department of Biology, Yeshiva University, 500 West W 185th Street, New York, NY, 10033, USA.
BMC Cancer. 2020 Jun 18;20(1):569. doi: 10.1186/s12885-020-07038-2.
KRAS mutations are prevalent in 40-45% of patients with colorectal cancer (CRC) and targeting this gene has remained elusive. Viruses are well known immune sensitizing agents. The therapeutic efficacy of oncolytic reovirus in combination with chemotherapy is examined in a phase 1 study of metastatic CRC. This study evaluates the nature of immune response by determining the cytokine expression pattern in peripheral circulation along with the distribution of antigen presenting cells (APCs) and activated T lymphocytes. Further the study evaluates the alterations in exosomal and cellular microRNA levels along with the effect of reovirus on leukocyte transcriptome.
Reovirus was administered as a 60-min intravenous infusion for 5 consecutive days every 28 days, at a tissue culture infective dose (TCID) of 3 × 10. Peripheral blood mononuclear cells (PBMC) were isolated from whole blood prior to reovirus administration and post-reovirus on days 2, 8, and 15. The expression profile of 25 cytokines in plasma was assessed (post PBMC isolation) on an EMD Millipore multiplex Luminex platform. Exosome and cellular levels of miR-29a-3p was determined in pre and post reovirus treated samples. Peripheral blood mononuclear cells were stained with fluorophore labelled antibodies against CD4, CD8, CD56, CD70, and CD123, fixed and evaluated by flow cytometry. The expression of granzyme B was determined on core biopsy of one patient. Finally, Clariom D Assay was used to determine the expression of 847 immune-related genes when compared to pre reovirus treatment by RNA sequencing analysis. A change was considered if the expression level either doubled or halved and the significance was determined at a p value of 0.001.
Cytokine assay indicated upregulation at day 8 for IL-12p40 (2.95; p = 0.05); day 15 for GM-CSF (3.56; p = 0.009), IFN-y (1.86; p = 0.0004) and IL-12p70 (2.42; p = 0.02). An overall reduction in IL-8, VEGF and RANTES/CCL5 was observed over the 15-day period. Statistically significant reductions were observed at Day 15 for IL-8 (0.457-fold, 53.3% reduction; p = 0.03) and RANTES/CC5 (0.524-fold, 47.6% reduction; p = 0.003). An overall increase in IL-6 was observed, with statistical significance at day 8 (1.98- fold; 98% increase, p = 0.00007). APCs were stimulated within 48 h and activated (CD8 CD70) T cells within 168 h as determine by flow cytometry. Sustained reductions in exosomal and cellular levels of miR-29a-3p (a microRNA upregulated in CRC and associated with decreased expression of the tumor suppressor WWOX gene) was documented. Reovirus administration further resulted in increases in KRAS (33x), IFNAR1 (20x), STAT3(5x), and TAP1 (4x) genes after 2 days; FGCR2A (23x) and CD244 (3x) after 8 days; KLRD1 (14x), TAP1 (2x) and CD244(2x) after 15 days. Reductions (> 0.5x) were observed in VEGFA (2x) after 2 days; CXCR2 (2x), ITGAM (3x) after 15 days.
Reovirus has profound immunomodulatory properties that span the genomic, protein and immune cell distribution levels. This is the first study with reovirus in cancer patients that demonstrates these multi- layered effects, demonstrating how reovirus can function as an immune stimulant (augmenting the efficacy of immuno-chemo-therapeutic drugs), and an oncolytic agent. Reovirus thus functions bimodally as an oncolytic agent causing lysis of tumor cells, and facilitator of immune-mediated recognition and destruction of tumor cells.
KRAS 突变在 40-45%的结直肠癌（CRC）患者中普遍存在，而针对该基因的靶向治疗仍然难以实现。病毒是众所周知的免疫致敏剂。在转移性 CRC 的 1 期研究中，检查了溶瘤性呼肠孤病毒联合化疗的治疗效果。本研究通过测定外周循环中细胞因子的表达模式以及抗原提呈细胞（APC）和活化 T 淋巴细胞的分布，来评估免疫反应的性质。此外，研究还评估了外泌体和细胞微 RNA 水平的变化，以及呼肠孤病毒对白细胞转录组的影响。
将呼肠孤病毒作为 60 分钟的静脉输注，连续 5 天，每 28 天一次，组织培养感染剂量（TCID）为 3×10。在给予呼肠孤病毒之前和之后的第 2、8 和 15 天，从全血中分离外周血单核细胞（PBMC）。在 PBMC 分离后，使用 EMD Millipore 多plex Luminex 平台评估血浆中 25 种细胞因子的表达谱。在给予呼肠孤病毒前后，测定外泌体和细胞 miR-29a-3p 的水平。用荧光标记的抗体对 CD4、CD8、CD56、CD70 和 CD123 染色外周血单核细胞，固定后通过流式细胞术进行评估。对一名患者的核心活检进行了颗粒酶 B 的表达检测。最后，通过 RNA 测序分析，与治疗前相比，使用 Clariom D 检测 847 个免疫相关基因的表达。如果表达水平翻倍或减半，则认为发生了变化，并且在 p 值为 0.001 时确定其显著性。
细胞因子测定表明，第 8 天 IL-12p40 上调（2.95;p=0.05）；第 15 天 GM-CSF（3.56;p=0.009）、IFN-y（1.86;p=0.0004）和 IL-12p70（2.42;p=0.02）上调。在 15 天的时间内，IL-8、VEGF 和 RANTES/CCL5 的总体水平下降。第 15 天，IL-8（0.457 倍，53.3%下降;p=0.03）和 RANTES/CC5（0.524 倍，47.6%下降;p=0.003）的减少具有统计学意义。IL-6 总体水平升高，第 8 天的统计学意义（1.98 倍;98%增加;p=0.00007）。48 小时内刺激 APC，168 小时内激活 CD8 CD70 活化 T 细胞，通过流式细胞术进行确定。持续记录到外泌体和细胞 miR-29a-3p 水平降低（miR-29a-3p 在 CRC 中上调，与肿瘤抑制基因 WW0X 的表达降低有关）。呼肠孤病毒给药后，KRAS（33 倍）、IFNAR1（20 倍）、STAT3（5 倍）和 TAP1（4 倍）在 2 天后增加;FGCR2A（23 倍）和 CD244（3 倍）在 8 天后增加;KLRD1（14 倍）、TAP1（2 倍）和 CD244（2 倍）在 15 天后增加。2 天后 VEGFA（2 倍）减少;CXCR2（2 倍），ITGAM（3 倍）在 15 天后减少。
呼肠孤病毒具有广泛的免疫调节特性，跨越基因组、蛋白质和免疫细胞分布水平。这是在癌症患者中使用呼肠孤病毒的第一项研究，证明了这种多层次的效应，展示了呼肠孤病毒如何作为免疫刺激剂（增强免疫化学治疗药物的疗效）和溶瘤剂发挥作用。呼肠孤病毒因此具有双重作用，既是溶瘤剂，能导致肿瘤细胞裂解，又是免疫介导的肿瘤细胞识别和破坏的促进剂。
