Characterization of microbial and host requirements for colonic biofilm assembly and biofilm-mediated colon tumorigenesis

散发性结直肠癌（CRC）作为癌症的主要原因之一，仍然是全球健康负担 和癌症相关的死亡，而且，引人注目的是，也正在成为一个严重的健康危机，在年轻的病人， 美国50岁以下。这些数据表明，新的努力，以确定贡献者的变化， 目前迫切需要建立一个区域中心景观。在这个R00提案中，我们试图剖析大会， 肿瘤发生机制背后的侵入性，多微生物结肠生物膜，这是以前 在Sears实验室中被确定为接近肝脏的CRC肿瘤的几乎普遍特征， 弯曲生物膜也在一个亚组（10 - 15%）的无CRC的健康患者中鉴定，其中 生物膜与早期的前致癌变化相关，这表明细菌生物膜在结直肠癌中发挥作用 发展这些生物膜也与多胺代谢物的质量升高有关。 基于光谱的分析，并通过一种方法在小鼠中诱导强烈的白细胞介素-17（IL-17）应答。 周，与人CRC预后不良相关的细胞因子特征。组织浆，由 人类生物膜在远端小鼠结肠中重新组装成生物膜;这些浆液中的一些（但不是全部） 在无菌ApcMin/+小鼠中直接致瘤10 - 15周。广泛培养一个 接种到小鼠体内后的致瘤浆液显示35种细菌分离株，当再次接种时， 在另外的小鼠中，重现了原始患者肿瘤浆液的肿瘤发生。我们的具体目标 在这些初步研究的基础上继续努力。特定目标（SA）1将确定关键细菌菌种 在35个分离物混合物中，它们负责生物膜形成和肿瘤发生。SA2将测试 假设IL-17和其他免疫参数是生物膜形成的关键介质， 生物膜介导的肿瘤发生，利用单细胞RNA测序，流式细胞术， qRT-PCR。使用IL-17抗体或敲除小鼠的治疗性调节将用于确认IL-17抗体在小鼠体内的表达。 IL-17在生物膜形成和生物膜介导的肿瘤发生中的需求。SA3将利用先进的 测定患者和小鼠中与生物膜相关的代谢物的质谱法 组织和这些代谢物在选定样品中的定位。该提案中的实验将 提供关于生物膜形成的宿主和微生物要求的有价值的信息， 生物膜介导的肿瘤发生，可能导致潜在的治疗靶点或生物膜的生物标志物。

Sporadic colorectal cancer (CRC) remains a global health burden as one of the leading causes of cancer and cancer-related deaths, and, strikingly, is also emerging as an acute health crisis in younger patients in the US under the age of 50. These data suggest that novel efforts to identify contributors to the changing landscape of CRC are urgently needed. In this R00 proposal, we seek to dissect the assembly and tumorigenic mechanisms behind invasive, polymicrobial colonic biofilms, which were previously established in the Sears laboratory to be a nearly universal feature of CRC tumors proximal to the hepatic flexure. Biofilms were also identified on a subset (10-15%) of healthy patients without CRC, in whom biofilms were associated with early, procarcinogenic changes, positing a role for bacterial biofilms in CRC development. These biofilms were also associated with elevations in polyamine metabolites in mass spectrometry-based analyses and induced a strong Interleukin-17 (IL-17) response in the mice by one week, a cytokine signature associated with a worse prognosis in human CRC. Tissue slurries made from human biofilms re-assembled into biofilms in the distal mouse colon; some - but not all – of these slurries were directly tumorigenic in germ-free ApcMin/+ mice by 10-15 weeks. Extensive culturing of one tumorigenic slurry following inoculation into mice revealed 35 bacterial isolates that, when re-inoculated into additional mice, recapitulated the tumorigenesis of the original patient tumor slurry. Our specific aims seek to build on these preliminary studies. Specific Aim (SA) 1 will identify the critical bacterial species within the 35 isolate mixture that are responsible for biofilm formation and tumorigenesis. SA2 will test the hypothesis that IL-17 and other immune parameters are critical mediators of biofilm formation and biofilm-mediated tumorigenesis utilizing a combination of single-cell RNA sequencing, flow cytometry, and qRT-PCR. Therapeutic modulation with an IL-17 antibody or knockout mice will be used to confirm the requirement of IL-17 in biofilm formation and biofilm-mediated tumorigenesis. SA3 will utilize advanced mass spectrometry methods to determine metabolites associated with biofilms in both patient and mouse tissues and the localization of these metabolites in select samples. The experiments in this proposal will provide valuable information regarding the host and microbial requirements for biofilm formation and biofilm-mediated tumorigenesis that may lead to potential therapeutic targets or biomarkers for biofilms.