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Intratumor heterogeneity may result from the evolution of tumor cells and their continuous interactions with the tumor microenvironment which collectively drives tumorigenesis. However, an appearance of cellular and molecular heterogeneity creates a challenge to define molecular features linked to tumor malignancy. Here we perform multiregional single-cell RNA sequencing (scRNA-seq) analysis of seven liver cancer patients (four hepatocellular carcinoma, HCC and three intrahepatic cholangiocarcinoma, iCCA). We identify cellular dynamics of malignant cells and their communication networks with tumor-associated immune cells, which are validated using additional scRNA-seq data of 25 HCC and 12 iCCA patients as a stable fingerprint embedded in a malignant ecosystem representing features of tumor aggressiveness. We further validate the top ligand-receptor interaction pairs (i.e., LGALS9-SLC1A5 and SPP1-PTGER4 between tumor cells and macrophages) associated with unique transcriptome in additional 542 HCC patients. Our study unveils stable molecular networks of malignant ecosystems, which may open a path for therapeutic exploration. Multiregion sequencing is needed to better capture the heterogeneity of hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA). Here, the authors analyse HCC and iCCA tumours with multiregion single-cell RNA-seq, revealing cellular dynamics and communication networks with immune cells.

SignificanceSingle-cell flow cytometry and genomic methods are rapidly increasing our knowledge of the diversity of cell types in metazoan tissues. However, suitably robust methods for placing these cells in a spatial context that reveals how their localization and putative interactions contribute to tissue physiology and pathology are still lacking. Here we provide a readily accessible pipeline (IBEX) for highly multiplex immunofluorescent imaging that enables a fine-grained analysis of cells in their tissue context. Additionally, we describe extensions of the IBEX workflow to handle hard-to-image tissue preparations and a method to facilitate direct integration of the imaging data with flow cytometry and sequencing technologies. The diverse composition of mammalian tissues poses challenges for understanding the cell–cell interactions required for organ homeostasis and how spatial relationships are perturbed during disease. Existing methods such as single-cell genomics, lacking a spatial context, and traditional immunofluorescence, capturing only two to six molecular features, cannot resolve these issues. Imaging technologies have been developed to address these problems, but each possesses limitations that constrain widespread use. Here we report a method that overcomes major impediments to highly multiplex tissue imaging. “Iterative bleaching extends multiplexity” (IBEX) uses an iterative staining and chemical bleaching method to enable high-resolution imaging of >65 parameters in the same tissue section without physical degradation. IBEX can be employed with various types of conventional microscopes and permits use of both commercially available and user-generated antibodies in an “open” system to allow easy adjustment of staining panels based on ongoing marker discovery efforts. We show how IBEX can also be used with amplified staining methods for imaging strongly fixed tissues with limited epitope retention and with oligonucleotide-based staining, allowing potential cross-referencing between flow cytometry, cellular indexing of transcriptomes and epitopes by sequencing, and IBEX analysis of the same tissue. To facilitate data processing, we provide an open-source platform for automated registration of iterative images. IBEX thus represents a technology that can be rapidly integrated into most current laboratory workflows to achieve high-content imaging to reveal the complex cellular landscape of diverse organs and tissues.

Spatial cellular context is crucial in shaping intratumor heterogeneity. However, understanding how each tumor establishes its unique spatial landscape and what factors drive the landscape for tumor fitness remains significantly challenging. Here, we analyze over 2 million cells from 50 tumor biospecimens using spatial single-cell imaging and single-cell RNA sequencing. We develop a deep learning-based strategy to spatially map tumor cell states and their surrounding environmental architecture, and find that different tumor cell states can be organized into distinct clusters, or “villages,” each supported by unique microenvironments. Notably, tumor cell villages exhibit village-specific molecular co-dependencies between tumor cells and their microenvironment and are associated with patient outcomes. Perturbation of molecular co-dependencies via random spatial shuffling of the microenvironment results in destabilization of the corresponding villages. We validate our findings using single-cell, spatial, and bulk transcriptome data from 740 liver cancer patients. This study provides insights into understanding tumor spatial landscape and its impact on tumor aggressiveness. Understanding how each tumor establishes its unique spatial landscape and what factors drive the landscape for tumor fitness remains significantly challenging. Here, the authors employ spatial single-cell imaging and single-cell RNA sequencing to analyze 50 liver cancer biospecimens and find that different tumor cell states may be organized into distinct clusters, or ‘villages’.

Background Adoptive cell therapy with tumor infiltrating lymphocytes is FDA approved for metastatic melanoma. TIL from patients with melanoma and factors relating to growth and reactivity have been studied; however, this has not been explored in patients with epithelial cancers. Patients and methods Metastatic epithelial tumors resected for TIL growth from 2014 to 2023 were analyzed. Two hundred and ninety-one operations were performed to collect TIL for potential treatment. Of these, 263 harvests were each processed for up to 24 individual fragment cultures and screened for neoantigen recognition of the expressed products of cancer mutations. Patient and tumor characteristics were collected. Endpoints were growth (defined as more than half of all fragment cultures expanded for viable cryopreservation) and patient-specific neoantigen reactivity (release of interferon-γ in cocultures measured by ELISpot and 4-1BB upregulation on flow cytometry). Results TIL fragments reached adequate growth for screening by 21 days. Metastatic resections from lung were more likely to grow TIL than all other resection sites combined (95%, p  = 0.0011), while hepatic resections were less likely to grow (69%, p  < 0.0001). One hundred and thirty-five patients (51%) had highly reactive TIL, 68 (26%) had weakly reactive TIL, and 60 (23%) had TIL with no neoantigen reactivity. Patients with prior exposure to immune checkpoint blockade therapy were less likely to have highly reactive TIL ( p  = 0.0325). Metastatic resection site impacted TIL reactivity against neoantigens, with those harvested from the lung more likely to show any reactivity (83%, p  = 0.0180), as well as high reactivity (59%, p  = 0.0066). Conclusions Prior immune checkpoint therapies reduced the likelihood of having highly reactive TIL. Neoantigen reactivity was more common in TIL from thoracic resections versus other sites. Conversely, hepatic lesions yielded TIL less likely to grow and with less reactivity. These results contribute to improved strategies for sequencing TIL with other therapies and planning TIL harvests for patients with epithelial cancers.

BackgroundOncolytic immunotherapy represents a unique therapeutic platform for the treatment of cancer. Here, we evaluated the safety and efficacy of the combination of pexastimogene devacirepvec (PexaVec) plus durvalumab (anti-programmed death ligand 1) with and without tremelimumab (anti-cytotoxic T-lymphocyte associated protein 4) in patients with standard chemotherapy refractory mismatch repair proficient (pMMR) metastatic colorectal cancer (mCRC) in a phase I/II trial.MethodsAdult patients with histologically confirmed advanced pMMR mCRC, who had progressed on at least two prior lines of systemic chemotherapy were studied in four cohorts. Patients received four doses of PexaVec IV at a dose of 3×108 plaque forming units (pfu) (dose level 1) or 1×109 pfu (dose level 2) every 2 weeks. Twelve days after the first PexaVec administration, patients received either 1500 mg of durvalumab every 28 days alone or an additional single dose of 300 mg tremelimumab on day 1. Responses were assessed every 8 weeks by CT or MRI. AEs were recorded. The primary endpoints were safety and feasibility. Secondary endpoints included progression-free survival (PFS) and overall survival. Paired tumor samples and peripheral blood were collected to perform immune monitoring.ResultsThirty-four patients with mCRC enrolled on to the study: 16 patients in the PexaVec/durvalumab cohorts and 18 patients in the PexaVec/durvalumab/tremelimumab cohorts. Overall, the combination of PexaVec plus immune checkpoint inhibitors did not result in any unexpected toxicities. Most common toxicities observed were fever and chills after PexaVec infusion. Two cases of grade 3 colitis, one case of a grade 2 myositis and one case of grade 3 hypotension resulted in discontinuation of immune checkpoint inhibitor and PexaVec treatment, respectively. The median PFS in the PexaVec/durvalumab/tremelimumab cohorts was 2.3 months (95% CI: 2.2 to 3.2 months) vs 2.1 months (95% CI: 1.7 to 2.8 months; p=0.57) in the PexaVec/durvalumab cohorts. Flow cytometry analysis of peripheral blood mononuclear cells revealed an increase in Ki67+CD8+ T cells on treatment.ConclusionPexaVec in combination with durvalumab and tremelimumab is safe and tolerable. No unexpected toxicities were observed. The combination of PexaVec/durvalumab/tremelimumab demonstrated potential clinical activity in patients with pMMR mCRC, but further studies are needed to identify the predictive biomarkers.Trial registration numberNCT03206073.

Wild-type KIT and PDGFRA gastrointestinal stromal tumors (GIST) are rare tumors with limited treatment options. We sought to determine the clinicopathologic features of wild-type GIST and identify factors that influence overall survival (OS) using a large national database. Retrospective evaluation of patients with wild-type GIST in the National Cancer Database (NCDB) was performed. Demographic, clinicopathologic, and treatment data were analyzed. Features associated with OS were investigated using Kaplan–Meier analysis and Cox proportional hazards model. 244 patients with median diagnosis age of 59 years (95% CI 57–63) were identified. The stomach was the most common primary site (57%) followed by the small intestine (35%). Surgical resection was performed on 85% of patients and 53% of patients received systemic therapy. Factors associated with decreased OS on multivariable analysis included small intestine primary (HR 2.72, 95% CI 1.13–6.69, P  = 0.026) and > 5 mitoses per 50 HPF (HR 4.77, 95% CI 1.86–13.2, P  = 0.001). Wild-type GISTs may be identified in older patients, with most arising in the stomach and small bowel. Surgery remains the principal treatment modality. Small intestine primary site and high mitotic count were associated with abbreviated OS.

Background Locally advanced gastric cancer (LAGC) presents a therapeutic dilemma, particularly as it often involves adjacent organs through desmoplasia or true pathologic invasion. To obtain a margin-negative resection, these tumors require en bloc gastrectomy with multivisceral resection (G+MVR), and contention remains regarding its safety and oncologic benefit. Methods We used the National Cancer Database to retrospectively evaluate the short- and long-term outcomes of patients with LAGC treated in the USA between 2004 and 2016. Associations with margin status and perioperative outcomes were calculated using logistic regression. Survival was estimated using Cox proportional hazards regression and the Kaplan-Meier method. Results Overall, 785 pathologic stage T4b (pT4b) patients diagnosed with LAGC underwent gastrectomy ( n = 438) or G+MVR ( n = 347). There was no association between G+MVR and short- or long-term mortality. Positive resection margins (HR 1.68, 95% CI 1.40–2.03), the presence of nodal disease (HRs 1.46–1.50), treatment at a high-volume center (HR 0.76, 95% CI 0.68–0.85), and the receipt of adjuvant chemotherapy (HR 0.64, 95% CI 0.51–0.80) were independently associated with overall survival. Diffuse-type histology was associated with higher rates of an R1 resection (OR 3.60, 95% CI 2.20–5.87). Perioperative and long-term survival metrics were comparable between patients with pT4a and pT4b LAGC who underwent a margin-negative G+MVR. Undergoing a margin-negative G+MVR imparted a 6-month survival benefit over non-curative gastrectomy alone ( p < 0.001). Conclusions Our study demonstrates the safety and long-term feasibility of G+MVR for disease clearance in well-selected patients with LAGC, and we advocate for their referral to high-volume centers for optimal care.

The liver connects the intestinal portal vasculature with the general circulation, using a diverse array of immune cells to protect from pathogens that translocate from the gut 1 . In liver lobules, blood flows from portal triads that are situated in periportal lobular regions to the central vein via a polarized sinusoidal network. Despite this asymmetry, resident immune cells in the liver are considered to be broadly dispersed across the lobule. This differs from lymphoid organs, in which immune cells adopt spatially biased positions to promote effective host defence 2 , 3 . Here we used quantitative multiplex imaging, genetic perturbations, transcriptomics, infection-based assays and mathematical modelling to reassess the relationship between the localization of immune cells in the liver and host protection. We found that myeloid and lymphoid resident immune cells concentrate around periportal regions. This asymmetric localization was not developmentally controlled, but resulted from sustained MYD88-dependent signalling induced by commensal bacteria in liver sinusoidal endothelial cells, which in turn regulated the composition of the pericellular matrix involved in the formation of chemokine gradients. In vivo experiments and modelling showed that this immune spatial polarization was more efficient than a uniform distribution in protecting against systemic bacterial dissemination. Together, these data reveal that liver sinusoidal endothelial cells sense the microbiome, actively orchestrating the localization of immune cells, to optimize host defence. The authors show that zonation extends to hepatic immune cells and that this spatial patterning is mediated by microbiome sensing by liver sinusoidal endothelial cells, and provide evidence that immune zonation is required to protect the host from the dissemination of blood-borne pathogens.

Background Altered expression of specific microRNAs (miRNA) is known to occur during colorectal carcinogenesis. However, little is known about the genome-wide alterations in miRNA expression during the neoplastic progression of primary colorectal cancers. Methods Using a miRNA array platform, we evaluated the expression of 668 miRNA in primary colonic adenocarcinomas. Biological functions of selected miRNA were evaluated with in vitro invasion assays. Results RNA was extracted for miRNA analysis from 65 primary colon cancers. We identified a seven-miRNA expression signature that differentiated stage I and stage IV primary colon cancers. We then demonstrated this signature was able to discriminate between stage II and III primary colon cancers. Six differentially expressed miRNA were downregulated in association with the development of metastases, and all 7 miRNA were complementary strand miRNA. We transfected HCT-116, a highly invasive colon cancer cell line, with corresponding downregulated miRNA and demonstrated that overexpression of three miRNA (miR200c*, miR143*, and miR424*) significantly abrogated invasive potential. Conclusion We have identified a seven-miRNA signature that is associated with metastatic potential in the primary tumor. Forced overexpression of three downregulated miRNA resulted in attenuation of in vitro invasion, suggesting direct tumor suppressive function and further supporting the biological importance of complementary strand miRNA.