The tumour-associated microbiota is an intrinsic component of the tumour microenvironment across human cancer types^1,2. Intratumoral host–microbiota studies have so far largely relied on bulk tissue analysis^1,2,3, which obscures the spatial distribution and localized effect of the microbiota within tumours. Here, by applying in situ spatial-profiling technologies⁴ and single-cell RNA sequencing⁵ to oral squamous cell carcinoma and colorectal cancer, we reveal spatial, cellular and molecular host–microbe interactions. We adapted 10x Visium spatial transcriptomics to determine the identity and in situ location of intratumoral microbial communities within patient tissues. Using GeoMx digital spatial profiling⁶, we show that bacterial communities populate microniches that are less vascularized, highly immuno‑suppressive and associated with malignant cells with lower levels of Ki-67 as compared to bacteria-negative tumour regions. We developed a single-cell RNA-sequencing method that we name INVADEseq (invasion–adhesion-directed expression sequencing) and, by applying this to patient tumours, identify cell-associated bacteria and the host cells with which they interact, as well as uncovering alterations in transcriptional pathways that are involved in inflammation, metastasis, cell dormancy and DNA repair. Through functional studies, we show that cancer cells that are infected with bacteria invade their surrounding environment as single cells and recruit myeloid cells to bacterial regions. Collectively, our data reveal that the distribution of the microbiota within a tumour is not random; instead, it is highly organized in microniches with immune and epithelial cell functions that promote cancer progression.

肿瘤相关微生物群是人类癌症类型的肿瘤微环境的固有组成部分^1,2。迄今为止，肿瘤内宿主微生物群研究主要依赖于大量组织分析^1,2,3，这掩盖了肿瘤内微生物群的空间分布和局部效应。在这里，通过将原位空间分析技术⁴和单细胞 RNA 测序⁵应用于口腔鳞状细胞癌和结直肠癌，我们揭示了宿主-微生物的空间、细胞和分子相互作用。我们采用 10x Visium 空间转录组学来确定患者组织内瘤内微生物群落的身份和原位位置。使用 GeoMx 数字空间分析⁶，我们发现，与细菌阴性肿瘤区域相比，细菌群落所填充的微生态位血管化程度较低、免疫抑制程度较高，并且与 Ki-67 水平较低的恶性细胞相关。我们开发了一种单细胞 RNA 测序方法，将其命名为 INVADEseq（侵袭粘附定向表达测序），通过将其应用于患者肿瘤，识别细胞相关细菌以及与其相互作用的宿主细胞，并揭示涉及炎症、转移、细胞休眠和 DNA 修复的转录途径的改变。通过功能研究，我们发现感染细菌的癌细胞以单细胞形式侵入周围环境，并将骨髓细胞招募到细菌区域。总的来说，我们的数据表明肿瘤内微生物群的分布不是随机的；相反，它在微生态位中高度组织化，具有促进癌症进展的免疫和上皮细胞功能。